Japanese B Encephalitis Virus Research Articles

Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs

The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

Development of antibody-array for detection of six arboviruses

Objective To develop an antibody-array system for multiple detection of antibodies against Japanese B encephalitis virus (JEV),Tick-borne encephalitis virus (TBV),Dengue virus ( DENV ),West Nile virus (WNV),Western equine encephalitis virus (WEEV) and East Equine encephalitis virus (EEEV).Methods Recombined antigens were spotted on array as capture antigens.Specific antibodies were detected by using a sandwich ELISA format.Rabbit antiserum was employed to select and confirm the specificity of antigens and to optimize the conditions of the assay.The detection efficiency of the system was validated by 40 clinical suspected serum samples and compared with the relative ELISA assays.Results Eleven recombined antigens were selected as diagnostic antigens with high specificity.Better detection could be achieved when scale of antigen concentrations were within 0.125-0.900 mg/ml and the serum dilutions were 1:100-1:1000.When detecting the 26 clinical suspected TBE serum samples,20 were IgG positive (76.9％),and 17 were IgM positive (65.3％) which was 96.1％ and 84.6％ consistent with the relevant ELLSA tests,the 8 clinical suspected JEV serum samples,4 were IgG positive (50.0％),and 5 were IgM positive (62.0％),which was 86.3％ and 90.1％ consistent with the relevant ELLSA tests.As for the 22 DEN serum samples,13 were IgG positive (60％) and 15 were IgM positive (68％) which was 85％ and 93％ consistent with ELISA.The specificity of the assay was 100％ and the sensitivity was higher than the relative ELISAs.Conclusion The developed antibody-array is highly specific and reliable,which could be used for the detection of antibodies against the 6 arboviruses. Key words: Arboviruses; Antibody-array; Detection

Epidemiology of Japanese–B– encephalitis infection in pigs in Riau and North Sumatera Provinces

Epidemiology study on Japanese-B-Encephalitis (JE) was conducted in Riau and North Sumatera Provinces. A total of 190 pig sera from Riau Province and 164 pig sera from North Sumatera were tested using competitive ELISA (C-ELISA) to detect antibodies against JE virus. Insect collection was also conducted using several methods near pig farms in those provinces and identified into species to gain more information on its role to distribute JE infection. Serological results indicated that 70% pig in Sumatera and 94% pig in Riau had antibodies against JE virus. The highest prevalence of reaktor was detected in pig of more than 4 months age in both Provinces. The results of insect collection showed that Culex tritaeniorchynchus and Culex quinquefasciatus were the most dominant species in both provinces. Based on serological testing, indicated that JE virus infected pig in Sumatera and Riau Provinces, and higher reactor was obtained in older pig. Culex tritaeniorchynchus and Culex quinquefasciatus were the dominant insect species in both provinces, hence those species had a possibility to play an important role of JE transmission. Key words: JE, pigs, serology, insects

The prevalence of Japanese-B-Encephalitis in different species in Indonesi

Japanese-B-Encephalitis (JE) is a zoonotic disease which is characterized by encephalitis, caused by JE virus. The situation of this disease has not been known in both animals and human in Indonesia. This paper reports serological finding using competitive - ELISA to evaluate 953 serum samples, comprised of chicken, ducks, cattle, goats, horses, dogs, pigs and human from different areas in Indonesia. The antibody against JE virus was detected in animals and human sera, with prevalence varied among species and location. Cattle showed the highest prevalence of reactor (51 %) while pigs, dogs and horses had the lowest reactor (11%,12% and ]4%). The highest prevalence of reactor in cattle was found in North Sumatera (86%) and the lowest was found in West Java (23%). In goat, the highest prevalence of reactor was found in West Kalimantan (59%) and the lowest was detected in South Sulawesi (14%). Antibody against JE virus was also detected in chicken with the highest prevalence in North Sumatera and West Kalimantan (44%) and the lowest was in South Sulawesi (36%). The highest percentage of reactor in pigs was detected in South Sulawesi (50%) and the lowest was detected in West Kalimantan (2%). In human, the highest prevalence of reactor was found in West Kalimantan (30%) and the lowest was fowld in Irian Jaya. This result provide more information for further research, therefor the JE cases in Indonesia and its social, economic and psychological impacts can be anticipated as earlyas possible. Key words : Japanese-B-Encephalitis, antibody, animals, human, ELISA

Establishment and Characterization of Japanese B Encephalitis Virus Persistent Infection in the Sf9 Insect Cell Line

Abstract.The Sf9 cell line, commonly used for gene expression by recombinant baculoviruses, can be productively infected by Japanese B encephalitis virus (JEV). Two wild-type JEV strains (P3 and SA14) caused a cytopathic effect (CPE) in the Sf9 cells, while no apparent CPE was caused by an attenuated strain (SA14–14–2). The JEV viral antigens were expressed in the infected Sf9 cells and intracellular virus particles were found by electron microscopy as a result of infection with all three strains. Titres of cell-associated and cell-free supernatant virus remained stable for relatively long periods of cultivation, suggesting that both wild-type and attenuated JEV strains established productive and persistent infections of Sf9 cells. The JEV produced by the Sf9 cells could be neutralized by anti-JEV reference serum, but relatively smaller plaques were formed in BHK21 cells infected with JEV that had been cultivated long term in Sf9 cells. This system for virus propagation has a number of potentially important uses for enhancing progress in JEV study and control.

An analysis of antigenic polypeptides of dengue viruses and their specificity with western blotting.

Dengue virus (DV) samples which harvested after having been either propagated in C6/36 cells or passaged by murine intracerebral inoculation were investigated by Western blotting. Two different denaturing methods with the same solution were selected before electrophoresis, one at 60 degrees C for 20 min; the other at 100 degrees C for 5 min. After the samples were heated at 60 degrees C for 20 min, 3 bands of sizes 46 ku, 70 ku and 98 ku were positively recognized by rabbit immunized sera. The peptide of 98 ku was DV group specific; While that of 46 ku could also react with the sera against both heterologous DV serotype and Japanese B encephalitis virus (JEV). After the samples were heated at 100 degrees C for 5 min, 3 peptides in size of 20 ku, 46 ku and 57 ku were recognized by the rabbit sera against both homologous and heterologous DV serotypes, while the peptide of 57 ku could also be recognized by anti-JEV serum. There was no significant difference in the results of Western blot between the DV antigens harvested from cellular culture and those from intra-cerebral inoculation.

Immunological Sequelae of Trichinella spiralis Infection in Mice: Effect on the Antibody Responses to Sheep Erythrocytes and Japanese B Encephalitis Virus

Immunosuppression of the antibody response to Japanese B encephalitis (JBE) virus and sheep erythrocytes (SRBC) was observed in mice infected with Trichinella spiralis. This suppression was paralleled by the presence of fewer antibody-forming cells to SRBC in the spleens of parasitized mice. Both primary and secondary complement-fixing antibody responses to JBE virus were suppressed, but the development of immunological memory was not affected. Parasitized mice demonstrated a normal blastogenic response to phytohemagglutinin and normal serum clearance rate of injected (125)I-labeled immunoglobulin G(2b), although the size of the extravascular fluid compartment was significantly increased. The data presented here and in previous reports suggest that sequential antigenic competition is a possible explanation for the humoral immunosuppression to heterologous antigens caused by T. spiralis infection.

Synergistic interaction of two agents in mice: Japanese B encephalitis virus and Trichinella spiralis.

A suitably timed pre-existing Trichinella spiralis infection greatly enhanced the intracerebral replication and mortality of peripherally inoculated Japanese B encephalitis (JBE) virus in mice. This increased mortality was associated with a decreased survival time and the effect was maximal when virus challenge occurred 7 days following infection with T. spiralis, and was absent by 21 days following helminthic infection. Titration of mouse sera and brains indicated that the increased virulence was paralleled by great increases in the ability of JBE virus to replicate in central nervous system (CNS) tissues. No evidence of increased duration or magnitude of JBE viremia was observed in T. spiralis-infected mice at any time. JBE virus killed normal and T. spiralis-infected mice equally upon intracerebral inoculation. It is suggested that T. spiralis infection may provide new routes of access for JBE virus into CNS tissues, or that the parasitic infection abrogates the host defense mechanism which normally aborts JBE virus replication in the brain. The applicability of these results to man is discussed.

Semi-commercial-scale production of Japanese B encephalitis virus vaccines from tissue culture.

This study was undertaken to modify and develop procedures for tissue culture-inactivated Japanese B encephalitis (JBE) virus vaccine production in large quantities. Various types of glass bottles were tried and, considering many advantages, long cylindrical roller (CR) bottles were selected. Several variables were investigated including number and volume of trypsinized cells to be seeded, volume of growth medium required for optimum cell growth, amount of calf serum, and volume of harvest medium for a high-titer virus yield. A good confluent cell sheet in CR bottles was obtained within a week by increasing the calf serum from 4 to 10% and when such tissue in a CR bottle was inoculated with 45,000 viral mean tissue culture infective doses directly into the medium, the cytopathological effects (CPE) appeared on day 5. High-titer virus yields were obtained when the harvests were made at 4(+) CPE using medium 199 with 2% human albumin at pH 8.3 to 8.5. No appreciable gain in titer was found from such harvests by blending to release intracellular virions. The production methods finally adopted gave consistently good results, and several inactivated JBE virus vaccine lots with minimum immunizing doses, ranging from 0.005 to 0.017 ml, were prepared using a large number of CR bottles in a simulated commercial-scale production system.

Semi-Commercial-Scale Production of Japanese B Encephalitis Virus Vaccines from Tissue Culture

This study was undertaken to modify and develop procedures for tissue culture-inactivated Japanese B encephalitis (JBE) virus vaccine production in large quantities. Various types of glass bottles were tried and, considering many advantages, long cylindrical roller (CR) bottles were selected. Several variables were investigated including number and volume of trypsinized cells to be seeded, volume of growth medium required for optimum cell growth, amount of calf serum, and volume of harvest medium for a high-titer virus yield. A good confluent cell sheet in CR bottles was obtained within a week by increasing the calf serum from 4 to 10% and when such tissue in a CR bottle was inoculated with 45,000 viral mean tissue culture infective doses directly into the medium, the cytopathological effects (CPE) appeared on day 5. High-titer virus yields were obtained when the harvests were made at 4(+) CPE using medium 199 with 2% human albumin at pH 8.3 to 8.5. No appreciable gain in titer was found from such harvests by blending to release intracellular virions. The production methods finally adopted gave consistently good results, and several inactivated JBE virus vaccine lots with minimum immunizing doses, ranging from 0.005 to 0.017 ml, were prepared using a large number of CR bottles in a simulated commercial-scale production system.

Studies of Arthropod-Borne Virus Infections in Chiroptera

Since the body temperature of the resting bat has been shown to parallel that of its environment over a wide range, these animals were used in a study to determine the effect of temperature on a virus under in vivo conditions. A strain of Japanese B encephalitis (JBE) virus was passed serially in big brown bats (Eptesicus f. fuscus) maintained at 24°C or 37° C, and the virus lines obtained were characterized and compared with lines developed by serial passage in hamster-kidney cell (HKC) cultures kept at the same temperatures. A virus line developed by passage in HKC cultures incubated at 24°C showed significant loss in virulence for mice, whereas passage of the virus in the bat maintained at 24°C did not result in any significant reduction in mouse pathogenicity. In addition, passage in HKC cultures at 37°C either did not affect or slightly increased the ability of the virus to multiply in this in vitro system at 37°C and 40°C, whereas passage in bats kept at 37°C, with body temperatures of 37°C or higher, seemed to de-adapt the virus for growth in HKC cultures at the higher temperatures. These observations suggest that the effect of temperature on certain characteristics of this strain of JBE virus varies depending on whether serial passage is carried out in vitro or in an in vivo system. The use of the bat as a host for the study of temperature-sensitive conditional-lethal mutants of arboviruses was discussed.

Bat Immunoglobulins Formed in Response to Experimental Japanese B Encephalitis (JBE) Virus Infection

Summary The immune response of the big brown bat (Eptesicus f. fuscus) to Japanese B encephalitis (JBE) virus was characterized in terms of the influence of the environmental temperature and the route of inoculation on the length of the antibody induction period and the types of immunoglobulins synthesized. Sucrose density gradient analyses, supported by data derived from DEAE-cellulose chromatography and immunoelectrophoresis, indicated that the bats produced at least two types of immunoglobulins. Hyperimmune bat plasma and hyperimmune guinea pig serum against JBE virus were employed in an examination of the virus-antibody interaction. The results are discussed in terms of the influence of the antibody response on the potential role of bats as reservoir hosts for arboviruses in nature.

1961～1965年茨城県における豚の日本脳炎抗体調査成績について

In the Ami area of Ibaraki Prefecture, swine sera were collected and examined for virus-neutralizing (VN), complenient-fixing (CF), and hemagglutination-inhibiting (HI) antibodies against Japanese B encephalitis (JBE) over five-month periods beginning in May every year from 1961 to 1965. In addition, similar antibody surveys were conducted on sera of small numbers of cattle, sheep, and goats in 1961 and 1962.1. In all the years, except 1963, VN and HI antibodies turned positive in the swine sera when August came. This result suggests that inapparent infection of JBE virus broke out among swine at the end of June, in the middle of July, or later. In 1963, those antibodies turned pbsitive in the swine sera in September.2. Antibodies turned positive in sera of cattle (in 1961 and 1962), sheep ad goats (in 1961) at such time as in swine sera. Ant ibody titers were generally lower in those sera than in swine sera.3. In 1963 when antibody-positive swine sera appeared later than in any other year, outbreaks of JBE among human beings were much fewer and later than in any other year. Atmospheric temperature was found lower in that year than any other year.

Analysis of the development of Japanese B encephalitis (JBE) virus: III. Electron microscope studies on inclusion bodies appearing in neurons and microglial cells infected with JBE virus

The fine structure of intracytoplasmic inclusion bodies seen in neurons and microglial cells from the thalamus of mice infected with JBE virus was studied in thin sections with the electron microscope: in many cases cells were examined in sets of sections cut in series. In the neuron fixed with the double fixation procedure, a roughly oval-shaped inclusion in the juxtanuclear region of the cytoplasm was delineated by a single-layered membrane or unit triple-layered membrane and contained spherical dense particles approximately 230 in diameter. In some infected microglial cells, the cytoplasm was almost entirely occupied by numerous inclusion bodies, the nucleus being replaced eccentrically by the bodies. The structures within the inclusion bodies were highly variable and they sometimes resembled, but were never identical with, cell organelles in the cytoplasm. It was assumed that the inclusion bodies seen in the microglial cells are products of phagocytosis since the cells are the counterparts of the macrophages of other parts of the body. It was possible to obtain information as to the presence of viral particles in the inclusion bodies by examining serial sections through neurons and microglial cells in the electron microscope. The particles, 210–250 in diameter, commonly seen within the inclusion bodies cannot be fully formed virus since mature virus particles are 370–380 in diameter.

Analysis of the development of Japanese B encephalitis (JBE) virus: II. Electron microscope studies of neurons infected with JBE virus

Analysis of the development of Japanese B encephalitis (JBE) virus: II. Electron microscope studies of neurons infected with JBE virus

INFECTIOUS RIBONUCLEIC ACID (RNA) OF JAPANESE B ENCEPHALITIS (JBE) VIRUS: HIGH YIELDS OF RNA AND ITS STABILIZATION.

SummaryH2O, 1.0 M Na2SO4, and 1.5 M MgSO4 protected the JBE virus from inactivation at 50°C, whereas 0.1 M and 1.0 M NaCI, 0.1 M Na2SO4 as well as 0.15 M MgSO4 inactivated the virus. However, the JBE-RNA was more stable in the presence of a high concentration of NaCl than in H2O and other salts tested. In addition, when EDTA was used either as a solution for homogenization of infected mouse brains or as a suspending solution of extracted infectious JBE-RNA, especially in the former case, the PFU of the RNA which were measured on chick embryo cell monolayers closely approached that of the virus and the infectivity of the RNA was protected from inactivation.

Analysis of the development of Japanese B encephalitis (JBE) virus: I. Electron microscope studies of microglia infected with JBE virus

Spherical particles with an average diameter of 375 were found in the neurons in medulla oblongata or thalamus of mice infected with Japanese B encephalitis virus. They were recognized to be similar to those encountered in the infected microglial cells reported previously. The virus particles were revealed within the vesicles or vacuoles of the cytoplasm, in the perinuclear space, and in the nucleus, but free neither in the cytoplasmic matrix nor in other cell organelles. The virus particles were different in the fundamentals of their structure depending on whether they were found in perikaryons or within myelinated axons: the former had no limiting membrane, but the latter were provided with a limiting membrane surrounding a dense nucleoid. The structure referred to as globular bodies represented the degenerated cytoplasmic matrix, which appeared being budded off from the cytoplasmic matrix into the vesicles.

Studies of Arthropod-Borne Virus Infections in Chiroptera

SummaryThe present report concerns the susceptibility of three species of insectivorous bats (Tadarida brasiliensis mexicana, Myotis lucifugus lucifugus, Eptesicus fuscus fuscus) to experimental infection with several strains of Japanese B encephalitis (JBE) and St. Louis encephalitis viruses. All three bat species proved to be susceptible to subcutaneous inoculation with one or more virus strains, the intensity of the infection depending on the mouse-passage level and origin of the strain. In general, high mouse-passage strains were less infective for bats than recent isolates. The characteristics of experimental arbovirus infection in bats include a persistent viremia (15–30 days) and viral invasion and multiplication in interscapular brown adipose tissue and, to a lesser degree, in brain and kidney tissue. Intracerebral inoculation of JBE virus produced a more intense and widespread infection in the big brown bat (Eptesicus f. fuscus) than subcutaneous inoculation, yet evidence of viral pathology was not observed in sections of infected brain or brown fat. None of the infected bats showed evidence of disease, indicating that these viruses are capable of replicating in various tissues of the bat without noticeably damaging cells or producing overt signs of encephalitis.Reasons why bats might be effective reservoirs of arboviruses in various phases of the year-round transmission cycles are discussed and the application of these experimental studies in the planning of field work is indicated.

Progress Report on Japanese B Encephalitis OCT-541 Attenuated Virus Strain

Last year we presented at these meetings a very brief report on the derivation and characteristics of an attenuated strain of Japanese B encephalitis (JBE) virus. This will be briefly summarized before presenting new work, including human trials. From Okayama Culex tritaeniorhynchus pool 541 (abbreviated OCT-541), known to contain JBE virus, we reisolated the virus directly in hamster kidney tissue culture (HKC), passed it serially in heavy inoculum by decreasing temperature steps, finally at 24°C. Then three serial plaque selections were made for particles of lowest mouse neurovirulence. Several pools grown at 24°C from this line proved to have exceedingly low neurovirulence for all laboratory animals by essentially all routes of inoculation, including more limited trials in monkeys and Mexican burros. Failure to develop antibody or immunity by almost all animals, except those inoculated intracerebrally with tremendous amounts of virus, suggested that this was too “cold” a strain and was essentially incapable of multiplying at the normal temperature of these animals.

螢光抗体法ならびに電子顕微鏡による日本脳炎ウイルスの研究及び日本脳炎のACTH療法

1) Purification of JBE (Japanese B encephalitis) virus and morphological demonstration of the virus particles were tried by means of fluorocarbon treatment and elcetron microscopyFluorocarbon extract of brains of mice infected with JBE virus revealed numerous spherical particles, 60 mμ in diameter by the shadowing method of electron microscopyElectron microscopic observation on ultrathin sections of chick red cells which were precipitated in hemagglutination induced with the fluorocarbon extract revealed round particles 30 to 40 mμ in diameter with an obscure nucleoid, on the surface of the red cells.These particles were believed to be JBE virus.2) In vivo locailzation of JBE virus antigens was persued by fluorescent antiody method in the brains of mice infected with JBE virus. The antibody was prepared from the serum of rabbits treated previously with the fluorocarbon extract.Specific fluorescence which was seen in granular forms was observed in nerve cells and partially in neuroglia cells of the substantia nigra and thalamus.3) JBE virus antigens were observed by fluorescent antibody method in phagocytes present in the cerebrospinal fluid of patients with Japanese B encephalitis. This finding was believed to contribute to early diagnosis of Japanese B encephalitis.4) JBE virus antigens were demonstrated by fluorescent antibody method in nerve cells and partially neuroglia cells of the brain obtained by autopsy from patients with Japanese B encephalitis.Specific fluorescence was seen in the Purkinje cells in the cerebellum, and granular layer of the cerebral cortex and basal ganglia, particularly substantia nigra.