Outbreak Of Japanese Encephalitis Research Articles

Isolation and genetic characterization of Japanese encephalitis virus from equines in India

Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E- and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.

Poly-γ-Glutamic Acid Nanoparticles and Aluminum Adjuvant Used as an Adjuvant with a Single Dose of Japanese Encephalitis Virus-Like Particles Provide Effective Protection from Japanese Encephalitis Virus

To maintain immunity against Japanese encephalitis virus (JEV), a formalin-inactivated Japanese encephalitis (JE) vaccine should be administered several times. The repeated vaccination is not helpful in the case of a sudden outbreak of JEV or when urgent travel to a high-JEV-risk region is required; however, there are few single-injection JE vaccine options. In the present study, we investigated the efficacy of a single dose of a new effective JE virus-like particle preparation containing the JE envelope protein (JE-VLP). Although single administration with JE-VLP protected less than 50% of mice against lethal JEV infection, adding poly(γ-glutamic acid) nanoparticles (γ-PGA-NPs) or aluminum adjuvant (alum) to JE-VLP significantly protected more than 90% of the mice. A single injection of JE-VLP with either γ-PGA-NPs or alum induced a significantly greater anti-JEV neutralizing antibody titer than JE-VLP alone. The enhanced titers were maintained for more than 6 months, resulting in long-lasting protection of 90% of the immunized mice. Although the vaccine design needs further modification to reach 100% protection, a single dose of JE-VLP with γ-PGA-NPs may be a useful step in developing a next-generation vaccine to stop a JE outbreak or to immunize travelers or military personnel.

Japanese Encephalitis, Tibet, China

Japanese Encephalitis, Tibet, China

Introduction of Japanese Encephalitis Virus Genotype I, India

Introduction of Japanese Encephalitis Virus Genotype I, India

Japanese Encephalitis in Hill and Mountain Districts, Nepal

To the Editor: Nepal, a landlocked country in Southeast Asia with an estimated population of 27 million, is divided administratively into 5 regions; 75 districts comprise 3 ecological zones that run from east to west. Altitude increases from south to north: the 20-district Terai plains in the south, the hill region in the center with 39 districts, and the 16-district mountain regions in the north. Japanese encephalitis (JE) is seasonally endemic to the Terai region, which borders the northern India states of Uttar Pradesh and Bihar. The first outbreak of JE in Nepal was reported in 1978 from the Terai district of Rupendehi (1). Since then, JE infection has been reported in animal reservoirs and in humans throughout the Terai region (1–5). Although few publications describe the presence of JE outside the Terai, an outbreak of JE in Kathmandu valley in the hill region was confirmed in 1997 (6), and a 2006 study reported JE endemicity in Kathmandu Valley (7). In recent years, the Ministry of Health and Population in Nepal has introduced public health interventions, including mass immunization campaigns, for JE prevention in these known JE-endemic areas.

Japanese Encephalitis Viruses from Bats in Yunnan, China

Genome sequencing and virulence studies of 2 Japanese encephalitis viruses (JEVs) from bats in Yunnan, China, showed a close relationship with JEVs isolated from mosquitoes and humans in the same region over 2 decades. These results indicate that bats may play a role in human Japanese encephalitis outbreaks in this region.

Curcumin Protects Neuronal Cells from Japanese Encephalitis Virus-Mediated Cell Death and also Inhibits Infective Viral Particle Formation by Dysregulation of Ubiquitin–Proteasome System

Japanese encephalitis (JE) is an arboviral disease common in Southeast Asia encompassing a population of 3 billion people. Periodic outbreak of JE takes hundreds of lives. Children are major victims of JE. About one third of JE patients die, and many of the survivors suffer from permanent neuropsychiatric sequel, owing to the lack of specific therapeutic measure. Curcumin is a naturally occurring phenolic compound extracted from Curcuma longa L. Previous studies have reported that curcumin possesses strong antioxidant, anti-inflammatory, antiviral activity. We used Neuro2a cell line and infected them with JE virus. The infected cells were treated with varying doses of curcumin. Cell viability, reactive oxygen species (ROS) production within the cells, and change in cellular membrane integrity were studied. The changes in expression of some signaling and stress-related proteins were also assessed. We also studied the inhibitory role of curcumin on the production of infective viral particles by dysregulation of the ubiquitin-proteasome system. In this study, we found that curcumin imparts neuroprotection in vitro, probably by decreasing cellular reactive oxygen species level, restoration of cellular membrane integrity, decreasing pro-apoptotic signaling molecules, and modulating cellular levels of stress-related proteins. We have also shown that curcumin, by inhibition of ubiquitin-proteasome system causes reduction in infective viral particle production from previously infected neuroblastoma cells.

Preventive strategies for frequent outbreaks of Japanese encephalitis in Northern India

Japanese encephalitis (JE) remains the most important cause of acute viral encephalitis and continues to spread to hitherto unaffected regions like Indonesia, Pakistan and Australia. Approximately 60% of the world population inhabits JE endemic areas. Despite its restricted range mostly in the developing countries,a high annual incidence of 50,000 cases and about 10,000 deaths has been reported. Disease can be fatal in 25% ases. Magnitude of the problem is even more alarming since the survivors are left with serious long-term neuropsychiatric sequelae. Almost every two years,epidemics of JE occur in Indian subcontinent with a high mortality. JE virus infection results in different disease manifestations in host from mild subclinical febrile illness to clinical infections leading to encephalitis. No antiviral treatment is so far available for JE. The prevention of JE can be achieved by controlling the vector or by immunization regime. The vector control in the rural areas,which are the worst affected ones,is practically almost impossible. Three vaccines that have been implicated against JE include inactivated mouse brain derived, inactivated cell culture derived and cell culture derived live attenuated JE vaccine. But each has its own limitation. Currently,attempts to synthesize recombinant DNA vaccine are being made. New therapeutics are on the way of development like use of minocycline, short interfering RNA, arctigenin, rosmarinic acid, DNAzymes etc. However,the immune mechanisms that lead to JE are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious JE vaccines.

The Landscape of New Vaccines for Japanese Encephalitis: Country-Level Strategies for Introduction

The first recorded major outbreak of Japanese encephalitis (JE) in Sri Lanka occurred in 1985–86 with 385 cases and 64 deaths in the North Central province. Outbreaks occurred in 1986–87 and 1987–88, the latter being the largest with 812 cases and 192 deaths in three adjoining districts. Cases occurred in rice cultivating areas with a network of irrigation canals supported by seasonal, moderate to heavy rainfall. Children aged 5– and young adults aged 20–24 years were predominantly affected. JE was also spreading to new areas with previously low transmission. To cope with this emerging challenge, the Ministry of Health’s Epidemiology Unit initiated phased JE immunization in 1988. Children aged 1–10 years were offered three primary doses and a booster of inactivated vaccine in the interpandemic period through a campaign approach. Over the years, JE incidence decreased as immunisation coverage increased. However, cases and occasional outbreaks were reported in other districts where immunization was not carried out, and the programme ultimately expanded to 18 districts.An increasing trend of adverse events following immunization with the inactivated JE vaccine threatened repercussions to the programme. Another obstacle was the increasing cost of the inactivated vaccine-US$4.50 per dose in 2006, a prohibitive factor to programme sustainability. Identifying an affordable, safe and immunogenic vaccine alternative was a high priority. The live, attenuated SA 14-14-2 JE vaccine (LJEV) appeared to be an appropriate, low cost, safe and potent alternative. With support from PATH, the Epidemiology Unit initiated a clinical trial in 2007 to ascertain the safety and immunogenicity of LJEV. Based on preliminary results, the government decided to introduce LJEV in place of the inactivated vaccine, hopefully expanding to routine EPI very soon. Currently, the Ministry of Health is negotiating a public sector price for procurement of LJEV and finalizing the recommended schedule. Cost savings from transitioning to LJEV will enable the introduction of new vaccines and will extend the JE immunization programme to adults in high endemic areas.

Clinical characteristics and laboratory assay of adult Japanese encephalitis patients in an outbreak in Yuncheng, Shanxi Province, 2006

To study the clinical and laboratory characteristics of adult Japanese encephalitis (JE) patients in a JE outbreak in Yuncheng, Shanxi Province in 2006. All the clinical data from the Second People's Hospital in Yuncheng city were analyzed, part of patients' sera and cerebrospinal fluid were tested by serology and RT-PCR. The majority of patients were middle-aged and elderly, 77.8% (35/45) of the total cases were more than 40 years old. Severe and fulminating type cases accounted for 60.0% (27/45). Most patients had underlying diseases. IgM antibody to JE virus (JEV) in serum was positive in each of the 45 patients analyzed and 4-fold or greater rise in sera neutralization antibody titer were found in convalescent serum. JEV nucleic acid was positive in part of cerebrospinal fluid specimens. Viral encephalitis emerged in Yuncheng city, Shanxi Province was Japanese encephalitis B, and most of the cases belonged to elderly group.

Mosquito Records from a Hot and Dry Climatic Area Experiencing Frequent Outbreaks of Japanese Encephalitis, Bellary District, Karnataka, India

Mosquito species occurring in Bellary district, Karnataka, India were surveyed for Japanese encephalitis (JE) and West Nile virus (WNV) from 2001 to 2003. A total of 37 mosquito species in 6 genera were recovered from larval and adult habitats. Aedes, Anopheles and Culex were represented by 11 species each, Mansonia by 2 species, and Armigeres and Lutzia by a single species. A total of 68,506 mosquitoes belonging to 20 species were collected at dusk. Most (74.6%) were Cx. tritaeniorhynchus and occurred in 2 peaks of abundance in February (304 per man hour density [PMHD]) and October (465 PMHD). The mosquito fauna of Bellary district is not diverse, possibly because of the hot and dry climatic conditions in the area.

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.

Japanese Encephalitis Outbreak, India, 2005

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.

Control of Japanese Encephalitis — Within Our Grasp?

The second half of 2005 saw one of the largest outbreaks of Japanese encephalitis in northern India in recent years. Dr. Tom Solomon writes that the best hope for controlling Japanese encephalitis lies in vaccination.

Reply to 'Encephalitis outbreak finds Indian officials unprepared'

We have found that the Japanese encephalitis outbreak in India, described in a recent news article1 by T.V. Padma, is overblown, leading to erroneous diagnosis of many encephalitis cases.

Should Vaccination be a Priority Approach for Japanese Encephalitis Prevention in Nepal ?

In Nepal a total number of 27584 Japanese encephalitis (JE) cases and 5382 deaths have been reported with an average case fatality rate of 19.5% in aggregate since 1978 to 2004. Past trend of the disease has shown that JE outbreaks are circumscribed and do not cover large areas. However it depends on the flight range of the vector mosquitoes presence of pigs as an amplifying hosts and the presence of a susceptible human population. During twenty six years period of time the highest number of cases were reported in 1997 (2953 cases) and the second highest were in 1999 (2924 cases). Similarly highest number of cases was reported from Far-western and Mid-western Development Regions. The lowest number of cases (54 cases) with mortality (CFR 29.6 percent) has been reported during the year 1981. The highest case fatality was observed in 1982 (CFR 46.3 percent). The overall mortality of JE varies from 8.5 percent (2004) to 46.3 percent (1982). (excerpt)

Population structure and dispersal of the freshwater mosquitoes Culex annulirostris and Culex palpalis (Diptera: Culicidae) in Papua New Guinea and northern Australia.

In 1995 and 1998, outbreaks of Japanese encephalitis (JE) virus occurred for the first time in Australia. Virus isolation from pools of mosquitoes indicated Culex annulirostris Skuse was the most likely vector. It was hypothesized that wind-blown mosquitoes introduced JE from Papua New Guinea to the Torres Strait and Cape York, northern Australia. This study used levels of genetic differentiation, as indirect evidence of dispersal of mosquitoes between the two continents. The results have demonstrated that in the region incorporating Western Province in PNG, the Torres Strait and Cape York Peninsula, Cx. annulirostris is represented by a panmictic population, indicating frequent widespread dispersal throughout the region of investigation. The closely related Cx. Palpalis Taylor was also present at some locations but the population structure of this species is uncertain. This supports the hypothesis that wind-blown Cx. annulirostris and possibly Cx. Palpalis could be a possible mechanism for introduction of JE virus into Australia.

Japanese encephalitis on Badu Island, Australia: the first isojation of Japanese encephalitis virus from Culex gelidus in the Australasian region and the role of mosquito host-feeding patterns in virus transmission cycles

During investigation of an outbreak of Japanese encephalitis (JE) in the Torres Strait, Australia, in 2000, mosquitoes were collected in Badu Island community and at a newly established communal piggery about 3 km from the community. A total of 94 285 mosquitoes, comprising 91 240 (96·8%) unengorged females, 1630 (1·7%) blood-engorged females and 1415 (1·5%) males, were processed for virus isolation. One isolate of JE virus was obtained from Culex gelidus, with a minimum infection rate of 12·4:1000. This is the first isolate of JE virus from Cx. gelidus in the Australasian region. No isolates were obtained from Cx. annulirostris, the primary implicated Australian JE vector. Analysis of mosquito host-feeding patterns, using gel diffusion, demonstrated that Cx. annulirostris and 5 other species fed predominately on mammals. Analysis of blood-fed mosquitoes collected within the community demonstrated that the proportion of Cx. annulirostris feeding on pigs in 2000 (2·3%) was significantly lower than that for the 1995–1997 period (31·3%). The removal of the pigs from Badu Island community has limited the contact between potential amplifying hosts and mosquitoes, thus potentially reducing the risk of transmission of JE virus to the human population.

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia.

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.

Molecular characterization of the first Australian isolate of Japanese encephalitis virus, the FU strain.

The complete genomic and predicted amino acid sequence of the Japanese encephalitis virus (JEV) FU strain, a human isolate recovered from the first outbreak of Japanese encephalitis in Australian territory, was determined. Comparison of the FU genome with 15 fully sequenced JEV genomes revealed high levels of sequence identity, ranging from 88.7% (GP78) to 89.7% (K94P05) for nucleotides and 96.8% (K94P05) to 98.0% (JaGAr01) for amino acid sequences. A total of 39 unique amino acid differences were found in the FU strain polyprotein. Phylogenetic analyses were performed on all available full-length JEV genomes and a selection of 64 E gene sequences from temporally and geographically diverse JEV strains. For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequences was also carried out. The FU strain was found to be most closely related to Korean isolate K94P05 in the full-length analysis and to Southeast Asian strains in the E and prM gene analyses. The E gene analysis corresponded well with the prM gene analysis and with previous genotyping studies using the prM gene. The epidemiological implications of this investigation are discussed.