A comparative analysis of Japanese Encephalitis Virus (JEV) in Asia: patterns of transmission, clinical diagnosis, and control strategies

The antigenic relationships between Japanese encephalitis (JE) virus and several other flaviviruses have been investigated using anti-JE virus monoclonal antibodies (MAbs). Seventeen MAbs directed against envelope protein E of JE virus were characterized and divided into eight MAb groups based on reactivity patterns in haemagglutination inhibition test, neutralization (N) test, ELISA and competitive binding assay with JE virus. The results suggest the existence of at least eight epitopes on the E protein of JE viruses. Analysis of cross-reactivity of the antibodies with several other flaviviruses indicated these findings JE virus, belonging to West Nile (WN) subgroup, is antigenically closely related to viruses in the same subgroup, i.e. Murray Valley encephalitis (MVE), WN and St. Louis encephalitis (SLE) viruses. Of these three viruses, JE virus has the closest relationship with MVE virus. WN virus is relatively close to JE virus, whereas SLE virus is the least closely related. Dengue viruses types 1 and 2, which belong to another subgroup of flaviviruses, show markedly less antigenic homology to JE virus. One of the critical N sites on the E protein showed JE virus specificity. Some cross-reactive antibodies which did not neutralize JE virus showed low but significant N activity against several other flaviviruses. Mixtures of several MAbs, which showed different reactivity patterns, potentiated the N activity against not only JE virus but also other members of the WN subgroup of flaviviruses, namely MVE, WN and SLE viruses.

Optimal conditions of radial haemolysis in gel (HIG) with Chikungunya (Chik) and Japanese encephalitis (JE) viruses were developed and compared with those of rubella virus. Sheep erythrocytes were found preferable for HIG with Chik and JE viruses, unlike rubella virus, where goose erythrocytes gave best results. Rooster erythrocytes were completely negative in the Chik and JE HIG, again differing from rubella virus, which was practically as good with rooster as with goose erythrocytes. Sheep erythrocytes with rubella virus gave incomplete reactions in HIG. Sensitization of erythrocytes with JE and Chik viruses is better, when borate-phosphate, and not phosphate buffer, is used. Optimum pH values for both viruses should be regarded. Sucrose-acetone, freon and borate-saline antigens from infected mice brain may be used for HIG with Chik and JE viruses.

The T lymphocytes play an important role in prevention and recovery from viral infections. To characterize T lymphocyte responses to Japanese encephalitis (JE) virus infections, we analyzed JE virus-specific T lymphocytes in peripheral blood mononuclear cells (PBMC) obtained from seven JE patients and 10 vaccinees who had received a formalin-inactivated, purified JE virus vaccine (Biken vaccine). These PBMC were examined for proliferative responses against live JE virus, a glutaraldehyde-fixed lysate of cells infected with JE virus, and extracellular particles (EPs; subviral membrane vesicles released from cells infected with recombinant vaccinia viruses encoding the JE virus premembrane and envelope proteins). Japanese encephalitis virus-specific T cell proliferation was demonstrated with PBMC from both patients and vaccinees after stimulation with infectious JE virus or the lysate of JE virus-infected cells. Proliferating PBMC included CD4+ T lymphocytes and CD8+ T lymphocytes in responses to either form of JE viral antigens. Responses to EPs were observed only with PBMC from some American vaccinees whose PBMC also responded to the virus and lysate. These results indicate that JE virus infection and immunization with an inactivated JE vaccine induce JE virus-specific CD4+ and CD8+ T memory lymphocytes that can be induced to proliferate by infectious JE virus and noninfectious JE antigens.

Tissue cultures of blood-sucking Arthropods and their use for the cultivation of Viruses and Rickettsiae

Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

Japanese Encephalitis, Tibet, China

Introduction of Japanese Encephalitis Virus Genotype I, India

Mosquito-borne flaviviruses with an enzootic transmission cycle like Japanese encephalitis virus (JEV) and West Nile virus (WNV) are a major public health concern. The circulation of JEV in Southeast Asia is well-documented, and the important role of pigs as amplification hosts for the virus is long known. The influence of other domestic animals especially poultry that lives in high abundance and close proximity to humans is not intensively analyzed. Another understudied field in Asia is the presence of the closely related WNV. Such analyses are difficult to perform due to the intense antigenic cross-reactivity between these viruses and the lack of suitable standardized serological assays. The main objective of this study was to assess the prevalence of JEV and WNV flaviviruses in domestic birds, detailed in chickens and ducks, in three different Cambodian provinces. We determined the flavivirus seroprevalence using an hemagglutination inhibition assay (HIA). Additionally, we investigated in positive samples the presence of JEV and WNV neutralizing antibodies (nAb) using foci reduction neutralization test (FRNT). We found 29% (180/620) of the investigated birds positive for flavivirus antibodies with an age-depended increase of the seroprevalence (OR = 1.04) and a higher prevalence in ducks compared to chicken (OR = 3.01). Within the flavivirus-positive birds, we found 43% (28/65) with nAb against JEV. We also observed the expected cross-reactivity between JEV and WNV, by identifying 18.5% double-positive birds that had higher titers of nAb than single-positive birds. Additionally, seven domestic birds (10.7%) showed only nAb against WNV and no nAb against JEV. Our study provides evidence for an intense JEV circulation in domestic birds in Cambodia, and the first serological evidence for WNV presence in Southeast Asia since decades. These findings mark the need for a re-definition of areas at risk for JEV and WNV transmission, and the need for further and intensified surveillance of mosquito-transmitted diseases in domestic animals.

BackgroundTo determine the prevalence and associated factors of seroprotection against Japanese encephalitis (JE) virus among HIV-infected adolescents stable on combination antiretroviral treatment (cART).MethodsA multicenter seroprevalence study was conducted in Thailand. Perinatally HIV-infected adolescents who aged 11–25 years, had previous evidence of severely immune suppression (CD4 < 15% or < 200 cells/mm3), were currently stable on cART (CD4 > 350 cells/mm3 for > 6 months or CD4 > 200 cells/mm3 with viral suppression [VS; plasma HIV RNA < 50 copies/mL] for > 12 months), and had completed a 3- or 4-dose series of mouse brain-derived inactivated JE vaccine (MBDV) during childhood were enrolled. Adolescents who had clinically or serologically confirmed recent JE virus infections, or received immunosuppressive agents or blood components within 6 months were excluded. Plaque reduction neutralization (PRNT50) assay was conducted to assess neutralizing antibodies to JE virus, and titers of ≥ 10 were considered seroprotective. Logistic regression analysis was performed to identify associated factors of JE seroprotection.ResultsOf 98 eligible adolescents, 54% were female, a median age was 19 years, and 11% were overweight. Ninety-five percent and 5% of adolescents received 3 and 4 doses of MBDV during childhood, respectively. A median duration since the last dose of MBDV was 16 years. At enrollment, 71% were on NNRTI-based cART regimens, a median cART duration was 13 years. A median current CD4 was 29%, and 89% had VS. Seroprotection against JE virus was identified in 28 (29%) adolescents; of whom, the geometric mean titer (GMT) of neutralizing antibody was 64 (95% CI: 39–106). Proportion of adolescents with JE seroprotection and GMT of neutralizing antibodies to JE virus slightly decreased over time after the last immunization (Figure 1). In a multivariable logistic regression analysis, seroprotection against JE virus was associated with younger age and greater current CD4 count (Table 1).ConclusionThe majority of our perinatally HIV-infected adolescents did not maintain seroprotection against JE virus although having completed a series of MBDV during childhood. JE revaccination is an important tool for disease prevention in these adolescents who live in JE endemic areas.DisclosuresAll authors: No reported disclosures.

Japanese Encephalitis Virus (JEV) is one of the most important endemic encephalitis in the world especially in Eastern and Southeastern Asia. JEV affects over 50,000 patients and results in 15,000 deaths annually [1]. JEV is the leading cause of viral encephalitis in 14 Asian countries. Approximately, 60% of the world’s population lives at risk in JEV-endemic regions of these countries. JEV is an acute, vector borne, noncontagious, and zoonotic viral infection of the central nervous system transmitted by the bite of infected mosquitoes [2]. JEV is a single stranded RNA virus and belongs to the family Flaviviridae [3]. JEV is transmitted between animals by mosquito species Culex, Anopheles, Mansonia [4]. Humans become infected with JEV coincidentally when living or travelling in close proximity to the enzootic cycle of the virus [4]. Encephalitis was first described in Japan in 1871, followed by outbreaks very few years. In 1924 the disease was named Japanese B encephalitis to distinguish it from von Economo’s encephalitis, or type A encephalitis (encephalitis lethargic); this was probably a form of autoimmune or post-infectious encephalitis, which caused an outbreak from 1916 to 1927. However, since then, the term “type A encephalitis” has not been used, and the terms Japanese B encephalitis has also fallen out to use [5]. Viral infection is maintained in enzootic cycles between birds and pigs. Water birds are the main reservoir for disseminating the JEV, whereas pigs are the amplifier hosts. Pigs usually do not show signs of the infection other than abortion and stillbirth, but they show continuous viremia, which allows the transmission of the virus to a human via mosquitoes. Humans and other large vertebrates including horses are not considered as efficient amplifying hosts and are, therefore “dead-end” hosts for the JEV [6]. The period of incubation is about 6-8 days, with a range of 4-15 days. JEV has a prodrome of a few days characterized by fever, headache, nausea, diarrhea, vomiting and myalgia, and they may last for several days. These symptoms are subsequently follows by altered mental status, which can range from mild confusion to coma. Headache and meningismus are more common in adults; seizures develop most often in children. Unintentional, rhythmic muscle movements are common, and mutism has been described as a presenting symptom. Sometimes, especially when there is an involvement of spinal anterior cells, JEV cause a syndrome of acute flaccid paralysis, resulting in a poliomyelitislike presentation. Generally fever disappears in about two weeks as long as with the other neurological symptoms [7]. Therapy for JEV infection is supportive. Different kinds of vaccines (inactivated, attenuated and chimeric) are available and used in several Asiatic countries. In Europe an inactivated vaccine is currently available. Another way to prevent Japanese encephalitis infection is to avoid mosquito bites in endemic rural areas more specifically close to irrigated rice fields and pig farms. Many mosquitoes are most active at dusk and dawn. People can use insect repellents when they are outdoors and wear long sleeves and trousers at these times, or consider staying indoors during these hours [8]. Sporadic introduction of JEV to new areas by migratory birds or by other ways may not necessarily lead to local viral circulation. JEV represents a human health threat in Europe merits further investigation [9]. The presence of JEV in Passeriformes of Tuscany constituting the first evidence of this antropo-zoonotic virus in Italy and also in Europe. The positivity to JEV of some house-sparrows confirms the potential epidemiological role of this bird in the Flavivirus Mosquito-borne JEV group spread, as observed for other viruses of JEV complex [10]. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed [11]. In order to increase surveillance in Europe improving diagnosis, monitoring and treatment of JEV different elements should be considered. First of all, at the present time there are no standard protocols on how to notify JEV infection and how “contact tracing” should be carry out thought different EU countries. For that reason education is extremely important, focusing especially on transmission pathways and preventive measures; it is also crucial to provide adequate resources to contrast the transmission of this disease. Moreover, in many countries at risk of infection, standard operating procedures and protocols for data exchange are not settled between human and animal health services. Therefore designing and creating a surveillance system for these vectortransmitted diseases should be targeted to develop common procedures and protocols for data exchange.

Japanese Encephalitis Virus: The Geographic Distribution, Incidence, and Spread of a Virus with a Propensity to Emerge in New Areas

Until 2010, no Japanese encephalitis (JE) had been reported from Delhi. Upon report of four confirmed cases of JE in September 2011, detailed investigations were carried out to determine whether the cases were imported or indigenous. Entomological surveys were carried out and all mosquito pools were tested for the detection of JE virus by ELISA method using specific monoclonal antibody. Human blood samples from contacts of the patients were tested by IgM-captured ELISA method. Pig's blood samples were also tested for the detection of JE virus. Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes were found. In contrast to rural areas, their breeding habitats were different in the city. 19 pools were tested. JE virus was detected in two pools of Cx.tritaeniorhynchus females reared from field-collected larvae, indicating vertical transmission. One pool of Cx.vishnui was also positive. This is the first report for the detection of JE virus in mosquitoes from Delhi. JE IgM antibodies in five contacts/residents indicate recent infection. JE virus was also detected in pigs. Present analysis shows that of four reported JE cases, three were confirmed indigenous, indicating that the virus is multiplying in the city. Mapping of infected JE vector mosquitoes in the cities is required for preventive measures to contain further spread of the disease.

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.

To the Editor: Japanese encephalitis (JE) virus is a flavivirus that causes about 15,000 deaths in Asia every year. Mosquitoes and swine play important roles in the spread of JE virus in the paradomestic environment, the former as a vector and the latter as an amplifier. In the 1950s, approximately 5000 cases, about 20% fatal, were reported yearly in Japan, but recently fewer than 10 cases are reported each year. Reasons for the marked decrease in JE in Japan include decreases in the number of rice fields which are habitats of vector mosquitoes (mostly Culex tritaeniorhynchus), subsequent decreases in the mosquito population, and increased distance between the habitats of people and swine. However, urban residents are still bitten by mosquitoes during the summer, with the exception of northern Japan. Furthermore, 50–100% of Japanese pigs younger than 8 months have antibodies against the JE virus in summer and autumn, indicating that most Japanese pigs can still be JE virus producers. The extremely low reported incidence of JE in human patients in Japan, despite the high prevalence of antibodies against the JE virus in swine and continued mosquito bites in humans, constitutes a paradoxical situation. To explain this paradox, we proposed a hypothesis that human JE viral infections still occur, but produce only mild symptoms. To test this hypothesis, we measured titers of the nonstructural protein-1 (NS1) antibody,1 which increases only with natural JE viral infection, and titers of the HI antibody, which increases with both vaccination and JE viral natural infection, in blood samples obtained from 50 volunteers and patients, aged 1–88 years, at Eijudo Clinic, located in an urban area of eastern Tokyo. Blood samples were collected for 2–3 years from each subject. Results are shown in Figure 1. The black bars indicate changes in NS1 antibody titers in each serum series, and the gray bars show changes in HI antibody concentration. The adjacent bars are results for a single person. The bars are arranged horizontally by subject age. The NS1 titers indicate that the incidence of JE viral infection has increased with subject age, whereas HI titers, which reflect vaccination, have slowly fallen with subject age. Two of 50 subjects showed rapid and marked increases in the NS1 titer. One was a 57-year-old woman who took a 3-day trip from June 30 through July 2, 2002, to Okinawa, a southern Japanese island where 3 American soldiers contracted JE with severe sequelae several years ago.2 The NS1 titer on June 21 was <1:10 but by July 21 had increased to 1:20 and remained high for 1 year. During this time the subject exhibited no symptoms. Another subject was a 85-year-old woman who had remained at home for several years. The NS1 titer was <1:10 on July 8, 2002, but the titers on July 15 and September 8, 2002, were 1:80. From July 8 through 15 she had a low-grade fever, but no infectious focus was identified.FIGURE 1.: Range of NS1 and HI antibody titers against Japanese encephalitis. The black bars indicate changes in NS1 antibody titers in each serum series, and the grey bars show changes in HI antibody concentrations. The adjacent bars are results for a single person. Sera collected from each person for 2–3 years.We have shown rising NS1 antibody titers with subject age in a series of blood samples obtained at a clinic in eastern Tokyo and described 2 subjects with rapidly increasing NS1 titers. These results and a previous report3,4 suggest that human JE viral infection remains prevalent in Japan, although symptoms can be mild or absent. Therefore, vaccination against JE remains extremely important, although the Japanese Government halted strong recommendation of routine JE vaccination in 2005.5 Teiichi Matsunaga, MD, PhD Eijudo ClinicKatsushika-ku, Tokyo, Japan Mizue Shoda, MSc Eiji Konishi, PhD Department of International Health Kobe Univ. Graduate Sch. of Health Sciences Kobe, Japan

During 1959 to 1965, continuous surveys for arbovirus from mosquitoes were carried out in Gunma Prefecture. Mosquitoes were collected at study sites by aspirators, sweeping nets, bait traps, and/or dry ice traps from May to October every year. Pools of female mosquitoes identified by species were tested for arboviruses by intracerebral inoculation of suckling mice, and following results were obtained : 1) 12 species of mosquitoes were collected in study sites. However, Japanese encephalitis (JE) virus was recovered from only Culex tritaeniorhynchus and Culex pipiens, and arboviruses other than JE virus were obtained from Culex tritaeniorhynchus and Aedes vexana. No virus was isolated from other species.2) About half of the isolates other than JE virus were identified as group A, Simbu or Bunyamwera groups. The remaining virues are still on the way of identification study, but they were assumed to be immunologically related neither Getah (group A) nor Akabane (Simbu group) viruses.3) Mosquito infection with JE virus was regularly limited to the period from July to September in this area, but some of the Non-JE viruses were isolated in early season before JE virus appeared in mosquitoes.4) It was clear that JE and Getah viruses were the prevalent viruses in Japan, because they had been recovered every summer. On the other hand, Akabane and Bunyamwera viruses might be incidentally introduced in the area recently.5) Details of the methods employed for virus isolation were described.