We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of discordant pairs (both 2%; P = 0.13 and P = 0.25, respectively). Comparison of qualitative final diagnostic interpretations for serum and plasma samples were not significantly different: only 1.5% (3 of 201 for Dengue/JEV IgM and Dengue IgG) and 2.0% (4 of 201; IgM and NS1) showed discordant pairs. These results demonstrate that plasma containing EDTA is suitable for use in these ELISAs.
Immunoglobulin M (IgM) and IgG antibodies to Japanese encephalitis virus (JEV) were detected in acute-phase cerebrospinal fluid (CSF) specimens from patients with acute encephalitis by using a solid-phase radioimmunoassay of the antibody capture type. Of 12 patients with JEV infections subsequently proven by hemagglutination inhibition serology, 11 had JEV IgM antibodies, as measured by antibody capture radioimmunoassay, in the first CSF specimen (geometric mean titer, 1:2,500) compared with 0 of 8 patients with acute encephalitis proven not to be due to JEV. Specific IgM anti-JEV activity (units per microgram) was greater in CSF than in parallel serum specimens in all 11 positive cases by more than fourfold on the average (range, 1.4 to 13). Among seven patients with broadly reactive hemagglutination inhibition seroresponses typical of persons previously exposed to other flaviviruses, six had high levels of JEV IgG antibodies (as measured by antibody capture radioimmunoassay) in their acute-phase CSF (geometric mean titer, 1:26,000), whereas in five patients experiencing their first flavivirus infection, JEV IgG antibodies measured by antibody capture radio-immunoassay were either absent (one patient) or weakly reactive (four patients; geometric mean titer, 1:3,200). Specific IgG anti-JEV activity was greater in CSF than in parallel serum specimens in eight of the nine positive cases measured (range, 1.3- to 24-fold). The antibody capture solid-phase immunoassay approach is well suited for detecting specific antibody activity in CSF.
Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field study in southern Vietnam, 155 cerebrospinal fluid (CSF) and 341 serum samples were collected from 111 children and 83 adults with suspected encephalitis. The JEV MAC DOT, performed on site, was scored visually from negative to strongly positive by two observers, and the results were compared subsequently with those of the standard IgM capture enzyme-linked immunosorbent assay. For the 179 patients with adequate specimens, the MAC DOT correctly identified 59 of 60 JEV-positive patients and 118 of 119 JEV-negative patients (sensitivity [95% confidence intervals], 98.3% [92.1 to 99.9%]; specificity, 99.2% [95.9 to 100.0%]; positive predictive value, 0.98; negative predictive value, 0.99). The MAC DOT also correctly identified three patients with dengue encephalopathy. Admission specimens were positive for 73% of JE patients. Interobserver agreement for MAC DOT diagnosis was excellent (kappa = 0.94). The JEV MAC DOT is a simple and reliable rapid diagnostic test for JE in rural hospitals.
Increasing virulence of Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES). Despite the availability of vaccine, JE/AES cases and deaths have become regular features in the different rural districts of West Bengal (WB) state, India, indicating either the partial coverage of vaccine or the emergence of new strain of JEV. Therefore, a study was undertaken to characterize and compare the complete envelope (E) protein gene based molecular changes/patterns of JEVs circulating in WB.
Total of 98 AES case-patients’ samples were tested to detect the presence of JEV specific immunoglobulin M (IgM) antibody by Mac-ELISA method. Only JEV IgM negative samples with a history of ≤3 days’ illness were screened for virus isolation and RT-PCR. E gene sequences of JEV isolates were subjected to molecular phylogeny and immunoinformatics analysis.
Present study confirmed JEV etiology in 39.7% and 29.1% of patients presenting ≤15 days’ febrile illness, as determined by Mac-ELISA and RT-PCR respectively. Phylogenetic analysis based on complete E gene sequences of JEV isolates showed the co-circulation of JEV genotype I (GI) with genotype III (GIII). This study also demonstrated that isolate-specific crucial amino acid substitutions were closely related to neurovirulence/neuroinvasiveness of JE. On the basis of immunoinformatics analysis, some substitutions were predicted to disrupt T-cell epitope immunogenicity/antigenicity that might largely influence the outcome of vaccine derived from JEV GIII SA14-14-2 strain and this has been observed in a previously vaccinated boy with mild JE/AES due to JEV GI infection.
Based on molecular evolutionary and bioinformatic approaches, we report evolution of JEV at a local level. Such naturally occurring evolution is likely to affect the disease profile and the vaccine efficacy to protect against JEV GI may demand careful evaluation.
Japanese encephalitis virus; Envelope protein gene; Molecular phylogeny; Genotype III; Genotype I; Homology modeling; Hydrophilicity; T-cell epitope; West Bengal
The proportion of laboratory-confirmed Japanese encephalitis (JE) virus (JEV) infections was compared to the number of JE cases reported on the basis of seasonality and the clinical symptoms of hospitalized patients in Guizhou Province, China, between April and November 2006. Of the 1,837 patients with reported JE, 1,382 patients in nine prefectures were investigated. JE was confirmed in 1,210 of 1,382 (87.6%) patients by a JEV-specific immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA), heminested reverse transcriptase PCR, and virus isolation. Two strains of JEV belonging to genotype 1 were isolated. Other viral pathogens responsible for encephalitis, including echovirus, mumps virus, herpes simplex virus, and cytomegalovirus, were identified in 67 of 172 (38.9%) JE-negative cases. On the basis of the distribution of the laboratory-confirmed JE cases from different hospitals according to the Chinese administrative division, which included hospitals at the provincial, city, county, and township levels, county hospitals detected the highest number of JE cases (81.8%), whereas township hospitals detected the smallest number of JE cases (1.4%). Provincial and city hospitals had the highest and lowest rates of accuracy of providing a clinical diagnosis of JE, as confirmed by laboratory testing (91.8% and 76.7%, respectively). This study demonstrates that laboratory confirmation improves the accuracy of diagnosis of JE and that an enhanced laboratory capacity is critical for JE surveillance as well as the identification of other pathogens that cause encephalitic syndromes with clinical symptoms similar to those caused by JEV infection.
Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People’s Democratic Republic (PDR) (Laos).
Japanese encephalitis (JE) virus is the leading cause of viral neurologic disease and disability in Asia. In the present study JE virus-specific IgM in serum and CSF from acute encephalitis syndrome (AES) patients, attending Assam Medical College and Hospital (AMC and H), Dibrugarh, Assam from 2007 to 2009 were detected and different epidemiological parameters namely age, season and vaccination campaign were enumerated.
Materials and Methods:
A cross-sectional study on patients with AES admitted in AMC and H, Dibrugarh, Assam was done during 2007 to 2009. The different epidemiological features were characterized depending on a pretested structured questionnaire called the clinical information form (CIF). Serum and CSF obtained were tested by a Panbio JE-Dengue IgM Combo ELISA kit and JEV Chex kit (Xycton).
A z-test was used for the statistical analytic assessment.
Detection rate of JE was 39.4%, 51.1%, and 51.3% in the years 2007, 2008, and 2009 respectively. Cases of JE increased in the age group more than 15 years in the district where the vaccination program was undertaken. This increase of cases from pediatric to adults is also statistically significant by the z-test (P<0.05).
There was an increase in AES cases and also JE cases from 2007 to 2009. JE also showed a seasonal variation with maximum cases in the months of July and August. Although vaccination campaigns with the live attenuated vaccine SA-14-14-2 have started and are protecting the under-15 children, there is a shift of disease pattern in the older population.
Epidemiological parameters; Japanese encephalitis; Vaccination
We conducted a prospective hospital based study from February 2009-April 2011 to identify the possible pathogens of central nervous system (CNS) infections in adults admitted to a tertiary referral hospital (Patan Hospital) in Kathmandu, Nepal. The pathogens of CNS infections were confirmed in cerebrospinal fluid (CSF) using molecular diagnostics, culture (bacteria) and serology. 87 patients were recruited for the study and the etiological diagnosis was established in 38% (n = 33). The bacterial pathogens identified were Neisseria meningitidis (n = 6); Streptococcus pneumoniae (n = 5) and Staphylococcus aureus (n = 2) in 13/87(14%). Enteroviruses were found in 12/87 (13%); Herpes Simplex virus (HSV) in 2/87(2%). IgM against Japanese encephalitis virus (JEV) was detected in the CSF of 11/73 (15%) tested samples. This is the first prospective molecular and serology based CSF analysis in adults with CNS infections in Kathmandu, Nepal. JEV and enteroviruses were the most commonly detected pathogens in this setting.
To examine the accuracy of the admission tourniquet test in the diagnosis of dengue infection among Lao adults.
Prospective assessment of the predictive diagnostic value of the tourniquet test for the diagnosis of dengue infection, as defined by IgM, IgG and NS1 ELISAs (Panbio Ltd, Australia), among Lao adult inpatients with clinically suspected dengue infection.
Of 234 patients with clinically suspected dengue infection on admission, 73% were serologically confirmed to have dengue, while 64 patients with negative dengue serology were diagnosed as having scrub typhus (39%), murine typhus (11%), undetermined typhus (12%), Japanese encephalitis virus (5%), undetermined flavivirus (5%) and typhoid fever (3%); 25% had no identifiable aetiology. The tourniquet test was positive in 29.1% (95% CI = 23.2–34.9%) of all patients and in 34.1% (95% CI = 27.0–41.2%) of dengue-seropositive patients, in 32.7% (95% CI = 23.5–41.8) of those with dengue fever and in 36.4% (95% CI = 24.7–48.0) of those with dengue haemorrhagic fever. Interobserver agreement for the tourniquet test was 90.2% (95% CI = 86.4–94.0) (Kappa = 0.76). Using ELISAs as the diagnostic gold standard, the sensitivity of the tourniquet test was 33.5–34%; its specificity was 84–91%. The positive and negative predictive values were 85–90% and 32.5–34%, respectively.
The admission tourniquet test has low sensitivity and adds relatively little value to the diagnosis of dengue among Lao adult inpatients with suspected dengue. Although a positive tourniquet test suggests dengue and that treatment of alternative diagnoses may not be needed, a negative test result does not exclude dengue.
dengue; tourniquet test; diagnosis; adult; Laos
We investigated the epidemiology and etiology of encephalitis at four tertiary hospitals in Bangladesh during 2003–2005. Patients who met a clinical case definition for acute encephalitis and had cerebrospinal fluid (CSF) pleocytosis were eligible for enrollment; a standardized sampling pattern was used to enroll eligible patients. Recent Japanese encephalitis virus (JEV) infection was defined by presence of IgM antibodies against JEV in CSF or serum. Twenty (4%) of 492 cases had laboratory evidence of recent JEV infection; two died. All JE cases occurred during May–December, and cases were identified among all age groups. All cases resided in rural areas. Fifteen patients were re-assessed 4–6 weeks after hospitalization; 5 (33%) patients had physical disabilities and 7 (47%) reported cognitive difficulties. Infection with JEV is clearly an etiology of encephalitis in Bangladesh. Population-based studies to quantify burden of disease could assess options for targeted immunization programs.
Japanese encephalitis (JE) was once epidemic in most areas of China, including Wuhan, a city located in the central part of China. The incidence of JE dramatically decreased due to nationwide immunization with the live attenuated JE virus (JEV) vaccine, and no JE cases were reported during 2005–2008 in Wuhan. In 2009 and 2010, 31 JE cases reoccurred in this area. In this study, we investigated the causes of JE recurrence.
Methods and Findings
All JE cases were laboratory-confirmed by detecting the JEV-specific IgM antibody with an IgM-capture enzyme-linked immunosorbent assay (ELISA). All patients were children between 2 months and 9 years of age with a median age of 2 years. Of the 31 cases, 9 had received one or two doses of the JEV vaccine, 11 had not been immunized previously with the JEV vaccine, and 11 had an unclear immunization history. Through reverse transcription polymerase chain reaction (RT-PCR), sequencing, and phylogenetic analysis, two new strains of JEV were isolated from Culex tritaeniorhynchus and identified as genotype 1 JEV, rather than genotype 3, which circulated in this area previously.
Vaccine failure or missed vaccination may have caused JE recurrence. Local centers for disease control and prevention need to improve immunization coverage, and the efficacy of the JE vaccine needs to be reevaluated in a population at risk for disease.
Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is the sole etiologic agent of Japanese Encephalitis (JE); a neurotropic killer disease which is one of the major causes of viral encephalitis worldwide with prime public health concern. JE was first reported in the state of West Bengal, India in 1973. Since then it is being reported every year from different districts of the state, though the vaccination has already been done. Therefore, it indicates that there might be either partial coverage of the vaccine or the emergence of mutated/new strain of JEV. Considering this fact, to understand the JEV genotype distribution, we conducted a molecular epidemiological study on a total of 135 serum/cerebrospinal fluid (CSF) samples referred and/or collected from the clinically suspected patients with Acute encephalitis syndrome (AES), admitted in different district hospitals of West Bengal, India, 2010.
JEV etiology was confirmed in 36/135 (26.6%) and 13/61 (21.3%) 2–15 days’ febrile illness samples from AES cases by analyzing Mac-ELISA followed by RT-PCR test respectively. Phylogenetic analysis based on complete envelope gene sequences of 13 isolates showed the emergence of JEV genotype I (GI), co-circulating with genotype III (GIII).
This study represents the first report of JEV GI with GIII, co-circulating in West Bengal. The efficacy of the vaccine (derived from JEV GIII strain SA-14-14-2) to protect against emerging JEV GI needs careful evaluation. In future, JE outbreak is quite likely in the state, if this vaccine fails to protect sufficiently against GI of JEV.
Acute encephalitis syndrome; Japanese encephalitis virus; Genotype I; Genotype III; West Bengal
Historically, Japanese Encephalitis virus (JEV) genotype III (GIII) has been responsible for human diseases. In recent years, JEV genotype I (GI) has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF) of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM) and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5′-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive- and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, leading to an acute encephalitis and damage to the central nervous system (CNS). The mechanism of JEV pathogenesis is still unclear. DNA microarray analyses have been recently employed to detect changes in host gene expression, which is helpful to reveal molecular pathways that govern viral pathogenesis. In order to globally identify candidate host genes associated with JEV pathogenesis, a systematic mRNA profiling was performed in spleens and brains of JEV-infected mice.
The results of microarray analysis showed that 437 genes in spleen and 1119 genes in brain were differentially expressed in response to JEV infection, with obviously upregulated genes like pro-inflammatory chemokines and cytokines, apoptosis-related proteases and IFN inducible transcription factors. And the significant pathways of differentially expressed genes are involved in cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, antigen processing and presentation, MAPK signaling, and toll-like receptor signaling, etc. The differential expression of these genes suggests a strong antiviral response of host but may also contribute to the pathogenesis of JEV resulting in encephalitis. Quantitative RT-PCR (RT-qPCR) assay of some selected genes further confirmed the results of microarray assay.
Data obtained from mRNA microarray suggests that JEV infection causes significant changes of mRNA expression profiles in mouse spleen and brain. Most of differentially expression genes are associated with antiviral response of host, which may provide important information for investigation of JEV pathogenesis and therapeutic method.
We evaluated performance of three commercial Japanese encephalitis virus (JEV) IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) kits with a panel of serological specimens collected during a surveillance project of acute encephalitis syndrome in India and acute meningitis and encephalitis syndrome in Bangladesh. The serum and cerebral spinal fluid specimens had been referred to the Centers for Disease Control and Prevention (CDC) for confirmatory testing. The CDC results and specimen classifications were considered the reference standard. All three commercial kits had high specificity (95–99.5%), but low sensitivities, ranging from 17–57%, with both serum and cerebrospinal fluid samples. Specific factors contributing to low sensitivity compared with the CDC ELISA could not be determined through further analysis of the limits and dilution end points of IgM detection.
Japanese encephalitis (JE) vaccination is the most effective measure for preventing JE disease. The live attenuated JE vaccine, which has shown good efficacy and safety, has been widely used in China.
We report four laboratory-confirmed JE cases detected in JE-endemic areas during the JE virus (JEV) transmission season, who all received a first dose of live attenuated JE vaccine within 2 weeks prior to the onset of illness. All cases presented with acute encephalitis and rapidly reduced consciousness. All cerebrospinal fluid (CSF) samples from the patients were positive for JEV-specific immunoglobulin M (IgM) antibodies, but viral isolation and polymerase chain reaction (PCR) detection of JEV were both negative.
It is difficult to identify a causal link between the disease and the vaccination, as the source of positive CSF JEV IgM antibodies might be natural JEV infection or possibly due to a traumatic lumbar puncture. Our observations highlight the need for public health officers and doctors to consider reasonable vaccination policies during the JE season. In addition, continued surveillance as well as thorough investigation of any events that occur after JE vaccination is necessary.
A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis – Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.
Japanese encephalitis, caused by the Japanese encephalitis virus, is the most prominent viral encephalitis in Asia. Three billion people live in endemic areas and at least 50,000 clinical cases occur each year, although reliable vaccines are available. Concerning the burden caused by this disease, more should be done to prevent it. Good and reliable diagnostics are one of the prerequisites for an effective fight against the virus, but it is nearly impossible to produce and evaluate an in-house assay according to high standard quality criteria as done for commercial tests. Only a few commercial assays are available and the thorough evaluation of these assays is of great importance for diagnostic laboratories. The sensitivity and specificity are statistical measures to assess the performance of a diagnostic assay. In this study the Euroimmun IgM indirect immunofluorescence test (IIFT) was compared to the Panbio and InBios IgM ELISAs. It showed a specificity of 95% and 100%, and a sensitivity of 86% and 93.8%, respectively. The specificity of the IgG IIFT in comparison to PRNT50 was 100% and the sensitivity was 93.8%. Overall, the IIFT showed a comparable performance and could be used as an alternative to the established assays.
Current diagnosis of human flaviviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Broad application of this assay is hindered by a lack of standardized human positive-control sera that react with the wide variety of flaviviruses that can cause human disease, e.g., dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV). We have created a human-murine chimeric antibody combining the variable regions of the broadly flavivirus cross-reactive murine monoclonal antibody (MAb) 6B6C-1 and the constant region of human IgM to produce a standardized reagent capable of replacing human positive-control sera in a MAC-ELISA for the diagnosis of all human flaviviral infections. The human-murine chimeric IgM antibody secreted from plasmid-transformed Sp2/0-Ag14 cells had a level of serological activity identical to that of 6B6C-1 as measured by ELISA, immunoblotting, and MAC-ELISA for multiple members of the flavivirus genus, including WNV, SLEV, YFV, DENV, and JEV.
Japanese encephalitis is a severe central nervous system (CNS) inflammatory disease caused by the mosquito-borne flavivirus, Japanese encephalitis virus (JEV). In the current study we have investigated the immune responses against JEV in mice lacking expression of the chemokine receptor CCR5, which functions in activation and chemotaxis of leukocytes during infection. We show that CCR5 serves as a host antiviral factor against Japanese encephalitis, with CCR5 deficiency markedly increasing mortality, and viral burden in the CNS. Humoral immune responses, which are essential in recovery from JEV infection, were of similar magnitude in CCR5 sufficient and deficient mice. However, absence of CCR5 resulted in a multifaceted deficiency of cellular immune responses characterized by reduced natural killer and CD8+ T cell activity, low splenic cellularity, and impaired trafficking of leukocytes to the brain. Interestingly, adoptive transfer of immune spleen cells, depleted of B lymphocytes, increased resistance of CCR5-deficient recipient mice against JEV regardless of whether the cells were obtained from CCR5-deficient or wild-type donor mice, and only when transferred at one but not at three days post-challenge. This result is consistent with a mechanism by which CCR5 expression enhances lymphocyte activation and thereby promotes host survival in Japanese encephalitis.
Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.
Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl β-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C6-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.
Infection with Japanese encephalitis virus (JEV) causes cerebral inflammation and stimulates inflammatory cytokine expression. Glial cells orchestrate immunocyte recruitment to focal sites of viral infection within the central nervous system (CNS) and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since immune cell infiltration is prominent, we investigated the production of a responding chemoattractant, RANTES (regulated upon activation, normal T-cell expressed and secreted), in response to JEV infection of glial cells. Infection with JEV was found to elicit the production of RANTES from primary neurons/glia, mixed glia, microglia, and astrocytes but not from neuron cultures. The production of RANTES did not seem to be directly responsible for JEV-induced neuronal death but instead contributed to the recruitment of immune cells. RANTES expression required viral replication and the activation of extracellular signal-regulated kinase (ERK) as well as transcription factors, including nuclear factor kappa B (NF-κB) and nuclear factor IL-6 (NF-IL-6). The induction of RANTES expression by JEV infection in glial cells needed the coordinate activation of NF-κB and NF-IL-6. Using enzymatic inhibitors, we demonstrated a strong correlation between the ERK signaling pathway and RANTES expression. However, JEV replication was not dependent on the activation of ERK, NF-κB, and NF-IL-6. Altogether, these results demonstrated that infection of glial cells by JEV provided the early ERK-, NF-κB-, and NF-IL-6-mediated signals that directly activated RANTES expression, which might be involved in the initiation and amplification of inflammatory responses in the CNS.
As noted in other flaviviruses, the envelope (E) protein of Japanese encephalitis virus (JEV) interacts with a cellular receptor and mediates membrane fusion to allow viral entry into target cells, thus eliciting neutralizing antibody response. The formation of the flavivirus prM/E complex is followed by the cleavage of precursor membrane (prM) and membrane (M) protein by a cellular signalase. To test the effect of prM in JEV biology, we constucted JEV-MuLV pseudotyped viruses that express the prM/E protein or E only. The infectivity and titers of JEV pseudotyped viruses were examined in several cell lines. We also analyzed the neutralizing capacities with anti-JEV sera from JEV-immunized mice. Even though prM is crucial for multiple stages of JEV biology, the JEV-pseudotyped viruses produced with prM/E or with E only showed similar infectivity and titers in several cell lines and similar neutralizing sensitivity. These results showed that JEV-MuLV pseudotyped viruses did not require prM for production of infectious pseudotyped viruses.
Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as an alternative to immunostaining. By this method, NS1 antigens contained in culture fluids from cells stably transfected with the NS1 and NS2A genes were captured by a rabbit anti-NS1 polyclonal antibody. Three nanograms per well of NS1 antigen, corresponding to 1:2 to 1:8 dilutions of the culture fluid, was sufficient for testing. ELISA values were obtained by a single-serum dilution (1:100), which correlated with ELISA titers obtained by an endpoint method. Under a tentative cutoff value (0.122) statistically calculated from NS1 antibody levels of horses in an area where JEV is not endemic, a high level of qualitative agreement (85.3%) was obtained between the ELISA and immunostaining methods. A significant correlation coefficient (0.799; P < 0.001) was also obtained between the two methods. Three experimentally infected horses seroconverted no later than 13 to 23 days postinfection, whereas 4 field horses infected during an epizootic remained positive for NS1 antibodies for at least 40 weeks. Our results indicate that the ELISA used here was sufficiently sensitive to detect subclinical infections in vaccinated equine populations.