Japanese Encephalitis Virus Strains Research Articles

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.

Japanese Encephalitis Outbreak, Yuncheng, China, 2006

To the Editor: Japanese encephalitis (JE) epidemics have occurred only in Asia. More than 50,000 cases of JE with ≈10,000 deaths have been reported since 1998 (1,2). The People’s Republic of China reported 5,104 cases and 214 deaths in 2005. Most of these deaths occurred in infants (3,4). During July and August 2006, an outbreak of viral encephalitis occurred in Yuncheng, Shanxi Province, People’s Republic of China. A total of 66 cases (1.32/100,000 population) were reported, including 19 deaths (case-fatality rate 28.8%). The cases had a widespread distribution over 9 counties and involved 37 towns and 61 administrative villages. The ratio of male-to-female patients was 1:0.89. A distinct clinical feature of this outbreak was the age distribution. More than 86% of the patients were >30 years of age, with only 10% of patients 50 years of age (5). We report serologic and virologic findings for the 2006 outbreak of viral encephalitis. Forty-six clinical specimens collected from 34 patients who had a diagnosis of viral encephalitis, including 33 serum samples and 13 cerebrospinal fluid (CSF) samples, were studied. All serum samples were screened for immunoglobulin M (IgM) to West Nile virus (WNV) by using the WNV IgM-capture ELISA kit (PanBio, Brisbane, Queensland, Australia) and for IgM to dengue virus or Japanese encephalitis virus (JEV) by using the JE-Dengue IgM Combination ELISA kit (PanBio). Results for JEV were confirmed by using the JE Virus IgM-Capture ELISA kit (Shanghai B & C Enterprise Development Co. Ltd, Shanghai, People’s Republic of China). WNV-specific or dengue virus–specific IgM was not detected in any samples. JEV-specific IgM was detected in 27 (80%) patients, which indicated recent JEV infections. The other 7 patients were negative for JEV by ELISA and reverse transcription–PCR (RT-PCR). Increases >4-fold in neutralizing antibodies were detected in acute- and convalescent-phase serum samples from 9 patients (10 serum pairs were collected during the outbreak). Attempts were made to detect virus in CSF of patients and in 2,400 mosquitoes. Mosquitoes (mainly Culex spp.) were collected in cow sheds and hog pens around houses and processed into pools of 100. Total RNA was extracted from CSF or mosquito homogenate by using the QIAamp viral RNA extraction kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s specifications. RT was performed by using Ready-To-Go-You Prime First Strand Beads (Amersham Pharmacia Biotech, Piscatawy, NJ, USA) and a seminested PCR to amplify 492-bp gene fragments of the premembrane (PrM) sequence of JEV by using the Takara LA Taq PCR kit (Takara Bio Inc., Shiga, Japan). The primers were derived from Ishikawa strain genome sequences (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB051292,term_id:12082323,term_text:AB051292}}AB051292). Primers PrMF: 5′-CGT TCT TCA AGT TTA CAG CAT TAG C-3′ (251–275), PrMR1: 5′-CGY TTG GAA TGY CTR GTC CG-3′ (724–743), and PrMR2: 5′-CCY RTG TTY CTG CCA AGC ATC CAM CC-3′ (901–925) were used. JEV PrM gene was amplified from CSF of 6 (46%) of 13 patients and 10 of 24 pools of mosquitoes by using the same seminested RT-PCR. To identify JEV genotype(s) involved in this outbreak, PCR products were sequenced. Eleven sequences (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:EF434264-EF434274,start_term:EF434264,end_term:EF434274,start_term_id:148925198,end_term_id:148925218}}EF434264-EF434274) were obtained from 6 patients and 5 pools of mosquitoes. The 11 sequences were compared phylogenetically with17 known JEV strains of the 4 recognized genotypes (classified on the basis of a 240-nt region of the prM gene). As shown in the Figure, the 11 sequences were those of JEV. Figure Phylogenetic analysis of Japanese encephalitis virus strains predicted from premembrane gene sequences. Neighbor-joining tree was generated by using MEGA 3.1 software (www.megasoftware.net) and rooted with Murray Valley encephalitis (MVE) virus sequence ... Further analysis showed that these 11 sequences can be grouped into genotypes I and III. Both genotypes were found in patient and mosquito samples, indicating that these genotypes co-circulated during this JE outbreak. JE has been endemic in Yuncheng for many years (6). A vaccine against JE (SA14–14–2) has been used in this area in infants, but not in adults. This might be 1 reason why a higher adult incidence was found in this outbreak. JEV genotype III had been the predominant genotype in previous years, but genotype I has been recently detected at increased frequencies (7–10). Detection of 2 JEV genotypes in 1 epidemic has not been reported. Whether simultaneous circulation of >1 genotype during an outbreak indicates a new type of emergence of JEV or that this has occurred and not been detected is unknown.

Construction of Human Fab (Y1/K) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.

Molecular epidemiological analysis of Japanese encephalitis virus in China

Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002-2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.

Immunologic Analysis Induced by DNA Vaccine Encoding E Protein of Beijing-1 Strain Derived from Japanese Encephalitis Virus

Objective: We have compared the gene expression and DNA immunization efficacy encoding prME and E proteins of a different strain (JaGAr-01) derived from Japanese encephalitis virus. This study aimed to construct a recombinant encoding E protein of the Beijing-1 strain derived from Japanese encephalitis virus and analyze the humoral, cellular and protective immunity induced by the above recombinant. Methods: The recombinant pJBE containing E (1,500 bps) gene from the Beijing-1 strain of Japanese encephalitis virus was constructed and then transfected into the HepG2 cell line by liposome fusion. The expression of E (about 53 kD) protein in transfected cells was analyzed by Western blot using a specific anti-JEV-E antibody. BALB/c mice were vaccinated with 3 µg of pJBE by the gene-gun technique. JaGAr-01 and Beijing-1 strains (10⁵ PFU/100 µl) of Japanese encephalitis virus were given to BALB/c mice by intraperitoneal injection 3 weeks after double DNA immunization with a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. A lactate dehydrogenase activity release test was used to examine cytotoxic T lymphocyte activity after double DNA immunization. Results: The expression of about 53 kD protein associated with pJBE was determined in transfected HepG2 cells with specific anti-JEV-E antibody. A higher level of neutralization antibodies and the cytotoxicity effect were induced with pJBE immunization using the gene-gun technique, and were similar to those induced with inactivated vaccine derive from the Beijing-1 strain of Japanese encephalitis virus. Balb/c mice immunized with pJBE survived the challenge with the different strains of Japanese encephalitis virus; however, Balb/c mice immunized with inactivated vaccine did not survive the challenge with the JaGAr-01 strain of Japanese encephalitis virus at all. Conclusions: DNA vaccine containing the E protein gene derived from Japanese encephalitis virus can provide not only better efficacy including humoral and cellular immunity, but also cross-protection against infection with homologous and heterologous Japanese encephalitis virus.

High antibody prevalence in an unconventional ecosystem is related to circulation of a low-virulent strain of Japanese encephalitis virus

Liu-Chiu islet, a relatively isolated ecosystem that is free of rice cultivation, has long considered free of Japanese encephalitis (JE). However, a new strain (T1P1) of JE virus was isolated from the mosquito, Armigeres subalbatus, and a rather broad distribution of neutralizing antibody has been reported on the islet, suggesting that the circulating virus could be an attenuated strain. In an assessment on 219 blood samples obtained from residents of Liu-Chiu islet, the positive rate of JEV-specific IgM antibodies decreased with age while that of neutralizing antibodies increased with age. Both antibodies were mainly responsive to the T1P1 strain since higher positive rates and titers of specific neutralizing antibodies are shown in this investigation. Importantly, the T1P1 strain is herein characterized as being broader in neutralizing virus strains, stable in genetic traits, and productive in Vero cells. Taken together, the JE virus strain endemically circulating on Liu-Chiu islet may have served as a natural form of a live-attenuated vaccine. As a result, it possibly can be utilized as a new and effective vaccine candidate in the future.

Preparation and characterization of the monoclonal antibodies against Japanese encephalitis virus.

To prepare mouse monoclonal antibodies (McAbs) against Japanese encephalitis virus (JEV)and evaluate their biological characteristics. McAbs against JEV were prepared by immunizing, fusing, cloning and screening. Their sensitivity, specificity, universality and neutralizing function were analyzed with ELISA, IFA, NT and Western blot. Titers of three McAbs against JEV were higher than 106. Three McAbs only reacted with JEV and not with other nine arboviruses. F12.37 could react with ten strains of JEV and sensitively detected ten replicating strains of viruses in BHK cell. The strains P3 and SH03-103 of JEV were neutralized by F12.37, its titers of protecting 50% cell were 3.2x105 and 105. Western blot showed that F12.37 reacted with envelop(E)protein of JEV. Three McAbs against JEV had high titer and good specificity. And F12.37 was very sensitive and universal in reacting with JEV, and neutralized JEV of Genotype I and Genotype .The binding site of F12.37 lays in E protein of JEV.

E sequence analysis of persistently infected mutant Japanese encephalitis virus strains

A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM 310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced (E61Tyr --> Asp, E219His --> Tyr, E384Val --> Glu, E418Pro --> Ala) in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement (E51Arg --> Ser, E61Tyr --> Asp, E83Lys --> Glu, E123Ser --> Arg, E209Arg --> Lys, E227Pro --> Ser, E276Asp --> Ser, E290Arg --> Lys, E387Lys --> Arg, E418Leu --> Pro, E454Arg --> Gly) was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 (Tyr --> Asp) may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

Comparison of nucleotide and deduced amino acid sequences of E gene of the newly isolated Japanese encephalitis virus strains and inactivated vaccine strain P3

To analyze the difference of nucleotides and deduced amino acids sequences E gene between the newly isolated Japanese encephalitis (JE) virus strains from mosquitoes or patients and P3 strain. The E gene sequences of corresponding strains of JE virus were obtained from GenBank. Computer analyze of nucleic acid data and deduced amino acid sequence were accomplished using the Clustal X (1.8), DNASTAR, GENEDOC (3.2) programs. The result showed that compared with the Fujian strains and P3 strain the nucleotide sequence homology was up to 98.3%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with the Shanghai strains and P3 strain, the nucleotide sequence differences were 12%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with P3 strain, there were nineteen amino acid variations in E gene of all the newly isolated strains. Between P3 and all the newly isolated JE virus strains, there are three common variations at E-129, E-222, E-366. And two common variations E-160 and E-487 were found only in Fujian strains, common variations at E-129, E-222, E-227, E-366 in Shanghai strains. There are some differences between P3 strain and JE viruses which were isolated from mosquitoes belonging to genotype I in Shanghai and from patients belonging to genotype III from Fujian province. But these variations are not in the important locations affecting the biological characteristic of the viruses.

A Single siRNA Suppresses Fatal Encephalitis Induced by Two Different Flaviviruses

BackgroundJapanese encephalitis virus (JEV) and West Nile virus (WNV) are neurotropic flaviviruses that can cause acute encephalitis with a high fatality rate. Currently there is no effective treatment for these infections.Methods and FindingsWe tested RNA interference (RNAi)-based intervention to suppress lethal JE and WN encephalitis in mice. To induce RNAi, we used either lentivirally expressed short hairpin RNA (shRNA) or synthetic short interfering RNA (siRNA). As target, we selected the cd loop-coding sequence in domain II of the viral Envelope protein, which is highly conserved among all flaviviruses because of its essential role in membrane fusion. Using as a target a species-specific sequence in the cd loop that is conserved only among the different strains of either JEV or WNV, we could achieve specific protection against the corresponding virus. However, by targeting a cross-species conserved sequence within the cd loop, we were able to protect mice against encephalitis induced by both viruses. A single intracranial administration of lentivirally delivered shRNA or lipid-complexed siRNA before viral challenge or siRNA treatment after viral challenge was sufficient for protection against lethal encephalitis.ConclusionsRNAi-based intervention affords near complete protection from both JEV- and WNV- induced encephalitis in mice. Our results show, to our knowledge for the first time, that siRNA can be used as a broad-spectrum antiviral agent for treating encephalitis caused by multiple related viruses.

Molecular characterization of attenuated Japanese encephalitis live vaccine strain ML-17

The Japanese encephalitis (JE) zoonotic vaccine strain ML-17 was sequenced and compared to related JE virus strains to identify genomic attenuation markers. Relative to its parental strain, JaOH0566, 25 nucleotide alterations and 10 amino acid changes to, prM/M(2), NS2A(1), NS4B(3) and NS5(4) proteins were recorded. Both structural-gene changes were in the prM/M region (127Met → Ile and 274Asn → Thr). To study the effects of these prM/M changes, mutants bearing the changes were prepared using an infectious clone of JaOArS982 previously established at this lab. Compared with JaOArS982, mutant 127(Met → Ile) showed marked reduction in murine neuroinvasiveness. Mutant 274(Asn → Thr), showed slight reduction. Neither mutant recorded ML-17-equivalent attenuation, implying that prM/M changes need to combine with other recorded genomic differences to cause attenuation. Importantly, ML-17 with its unchanged E region, presents a possible backbone candidate for preparation of “E-replacement” type live attenuated flavivirus chimeric vaccines.

Genotypic Analysis of Japanese Encephalitis Virus Strains Isolated from Swine in Tokyo, Japan

Genotypic Analysis of Japanese Encephalitis Virus Strains Isolated from Swine in Tokyo, Japan

Detection of West Nile virus and Japanese encephalitis virus using real-time PCR with a probe common to both viruses

A diagnostic method to distinguish between West Nile virus (WNV) and Japanese encephalitis virus (JEV) based on fluorogenic real-time polymerase chain reaction (TaqMan) assays was developed. To detect WNV and JEV with a single probe, a probe was designed to correspond to sequences in the core protein region that are shared by both viruses. The specificity of this assay depended on the primer sets used, which were specific to the target virus sequences: the primer set for WNV could detect only WNV strains and the primer set for JEV could detect only JEV strains. The assays were tested by detection of viruses from experimentally infected animal tissues. The method described in this study will be useful for the simultaneous discrimination of WNV and JEV in areas where JEV is endemic, such as East Asia.

Sensitive and specific detection of strains of Japanese encephalitis virus using a one-step TaqMan RT-PCR technique.

A rapid, sensitive, and accurate laboratory diagnostic test is needed for distinguishing Japanese encephalitis virus (JEV) from other diseases featuring similar clinical symptoms and also for preventing potential outbreaks. In this study, a TaqMan reverse transcription (RT)‐polymerase chain reaction (PCR) assay was developed for rapid detection and quantification of the viral RNA of various JEV strains. A consensus JEV NS3 region was chosen to design the primers and the TaqMan probe. The JEV TaqMan assay used the EZ‐rTtH RT‐PCR system featuring advantages such as a one‐step, high‐temperature RT reaction modality and preventing carry‐over contamination. The sensitivity of the JEV TaqMan assay for detecting in vitro‐transcribed JEV NS3 RNA was estimated to be one to five copies of RNA per reaction. For cultured JE virions, less than 40 plaque forming unit (PFU)/ml of virus load (corresponding to 0.07 PFU/test) could be detected. In addition, the JEV TaqMan assay could detect all seven strains of JEV tested, but provided negative results for nine other flaviviruses and encephalitis viruses tested. The JEV TaqMan assay demonstrated greater sensitivity and specificity than traditional RT‐PCR methods as has been previously reported. The application of the JEV TaqMan assay herein has been shown to the sensitive detection of the JEV from both mosquito pools and also JEV‐spiking human blood. The assay should be of use in diagnostic laboratory conduct and could be used to replace or complement time‐consuming viral‐culture methods, thus achieving more rapid, sensitive, and highly specific identification of JEV infection. J. Med. Virol. 74:589–596, 2004. © 2004 Wiley‐Liss, Inc.

Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses.

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.

Shift in Japanese encephalitis virus (JEV) genotype circulating in northern Vietnam: implications for frequent introductions of JEV from Southeast Asia to East Asia.

This study analyses the evolutionary relatedness of 16 Japanese encephalitis virus (JEV) isolates (nine from Vietnam and seven from Japan) to previously published JEV strains using E gene sequence data. Vietnamese and Japanese strains isolated between 1986 and 1990 were found to cluster in genotype 3. However, more recent Vietnamese and Japanese strains isolated between 1995 and 2002 grouped within genotype 1, now a dominant though previously unreported genotype in Vietnam. In addition, in this study, strains isolated between 1995 and 2002 were more closely related to those isolated in the 1990s than to the older genotype 1 strains. Recently, the introduction of JEV genotype 1 into Japan and Korea has also been reported. Hence this genotype shift phenomenon may be occurring throughout all East Asia. Further studies on JEV ecology are needed to clarify the mechanism of JEV genotype 1 spread to new territories.

Immunogenicity and protective efficacy in mice of a formaldehyde-inactivated Indian strain of Japanese encephalitis virus grown in Vero cells

P20778, an Indian strain of Japanese encephalitis virus (JEV) obtained from Vellore in the Southern India, was grown in Vero cells cultured on microcarriers in a spinner flask. The virus was formalin-inactivated and its immunogenicity and protective efficacy in mice were tested in comparison with a commercially available vaccine. Our studies indicated that formalin-inactivated JEV P20778 induced high levels of protective immunity in mice. Virus inactivation with formalin at 22 °C, which required shorter incubation period, was found to be as good or better to virus inactivation at 4 °C for generating high titers of anti-JEV antibodies. Similarly, the 22 °C-inactivated virus generated JEV neutralizing antibody titers as good or higher than those induced by the 4 °C-inactivated virus. Thus, for the vaccine production, inactivation of JEV with formalin at 22 °C would be a preferred method as it is faster and does not require cold room storage.

Protection against Japanese encephalitis virus strains representing four genotypes by passive transfer of sera raised against ChimeriVax™-JE experimental vaccine

The ability of antisera raised against a candidate Japanese encephalitis virus (JEV) vaccine, ChimeriVax™-JE, and the currently licensed vaccine, JE-VAX ®, to protect against strains of JEV representing the four major genotypes was assessed. Neutralization assays and passive protection studies in mice showed that greatest protection was provided against strains of genotypes II and III, although some protection was also afforded against genotypes I and IV strains. ChimeriVax™-JE stimulated protection that was comparable or superior to the JE-VAX ® control.

A model to study neurotropism and persistency of Japanese encephalitis virus infection in human neuroblastoma cells and leukocytes.

Japanese encephalitis (JE) is the most common mosquito-borne encephalitis in the Asia-Pacific region. Patients with JE usually present neuronal involvement, but other organ involvement is relatively rare. Employing human neuroblast-derived (NB) cell lines and different blood cells (erythrocytes, lymphocytes, granulocytes and monocytes), the neurotropism and persistency of Japanese encephalitis virus (JEV) in human cells was investigated. It was found that JEV could not replicate in erythrocytes, granulocytes or lymphocytes. Monocytes and NB cell lines could support replication of JEV as demonstrated by expression of viral NS3 antigen and virus plaque-forming units (p.f.u.). JEV could replicate more efficiently in neuroblastoma (HTB-11) cells than in monocytes after infection for 48 h (2.1+/-1.2x10(7) vs 2.8+/-0.7x10(2) p.f.u. ml(-1)). Two different strains of JEV revealed a similar infectivity to different leukocytes and four NB cell lines. In a kinetic study, it was found that JEV-infected monocytes possessed a high viability (90 %) after infection for 5 days, while JEV-infected neuroblastoma cells suffered cell apoptosis in 2 days and decreased viability to less than 1 % in 5 days. Further studies showed that monocytes could take up JEV rapidly, displaying a log scale increase of intracellular JEV titres in 9 h after infection. Significantly, extracellular production of JEV by monocytes started in 12 h, peaked in 3 days and persisted for more than 3 weeks. These results suggest that JEV-infected monocytes may play an important role in harbouring JEV for eventual transmission to NB cells and that modulation of JEV-induced NB cell apoptosis may be useful in treating patients with JE.

TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID(50) /ml and 11.2 TCID(50) /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (C(t)) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.