Origin and evolution of Japanese encephalitis virus in southeast Asia

Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens.     

Japanese Encephalitis��A Pathological and Clinical Perspective

Japanese encephalitis (JE) is the leading form of viral encephalitis in Asia. It is caused by the JE virus (JEV), which belongs to the family Flaviviridae. JEV is endemic to many parts of Asia, where periodic outbreaks take hundreds of lives. Despite the catastrophes it causes, JE has remained a tropical disease uncommon in the West. With rapid globalization and climatic shift, JEV has started to emerge in areas where the threat was previously unknown. Scientific evidence predicts that JEV will soon become a global pathogen and cause of worldwide pandemics. Although some research documents JEV pathogenesis and drug discovery, worldwide awareness of the need for extensive research to deal with JE is still lacking. This review focuses on the exigency of developing a worldwide effort to acknowledge the prime importance of performing an extensive study of this thus far neglected tropical viral disease. This review also outlines the pathogenesis, the scientific efforts channeled into develop a therapy, and the outlook for a possible future breakthrough addressing this killer disease.     

Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection ( p  < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.     

Modelling Japanese encephalitis virus transmission dynamics and human exposure in a Cambodian rural multi-host system

Japanese encephalitis (JE) is a vector-borne zoonosis and the leading cause of human viral encephalitis in Asia. Its transmission cycle is usually described as involving wild birds as reservoirs and pigs as amplifying hosts. JE is endemic in Cambodia, where it circulates in areas with low pig densities (<70 pigs per km²), and could be maintained in a multi-host system composed of pigs, but also poultry as competent hosts, and dogs, cattle and humans as non-competent hosts. We used a mathematical model representing Japanese encephalitis virus (JEV) transmission in a traditional Cambodian village that we calibrated with field data collected in 3 districts of Kandal province, Cambodia. First, R₀ calculations allowed us to assess the capacity of the epidemiological system to be invaded by JEV and sustain virus transmission in villages in the 3 districts, and we predicted human exposure at the epidemiological equilibrium, based on simulations. Changes in spatial density of livestock, in agricultural practices, and epizootics (e.g., African swine fever), can profoundly alter the composition of host communities, which could affect JEV transmission and its impact on human health. In a second step, we then used the model to analyse how host community composition affected R₀ and the predicted human exposure. Lastly, we evaluated the potential use of dog JE seroprevalence as an indicator of human exposure to JEV. In the modeled villages, the calculated R₀ ranged from 1.07 to 1.38. Once the equilibrium reached, predicted annual probability of human exposure ranged from 9% to 47%, and predicted average age at infection was low, between 2 and 11 years old, highlighting the risk of severe forms of JEV infection and the need to intensify child immunization. According to the model, increasing the proportion of competent hosts induced a decrease in age at infection. The simulations also showed that JEV could invade a multi-host system with no pigs, reinforcing the assumption of poultry acting as reservoirs. Finally, the annual human exposure probability appeared linearly correlated with dog seroprevalence, suggesting that in our specific study area, dog seroprevalence would be a good proxy for human exposure.     

Historical review and surveillance of Japanese encephalitis, Republic of Korea, 2002–2004

Japanese encephalitis virus (JEV), transmitted by culicine mosquitoes, is endemic throughout much of South‐East Asia, extending to the Korean Peninsula. The zoonotic cycle is from large water birds to culicine mosquitoes, with swine as an amplifying host and man as an incidental host. Culex tritaeniorhynchus, the primary JEV vector in the Republic of Korea, populations peak in late August through to early September when most cases of Japanese encephalitis (JE) are reported. Cx. tritaeniorhynchus were observed near the Demilitarized Zone in each of the years that mosquitoes were assayed for JEV. Each year that vector mosquitoes were assayed for JEV, minimum field infection rates (number of JEV positive mosquites/1000 Cx. tritaeniorhynchus assayed) ranged from 0.31 to 3.27. The epidemiology of JE has been recorded in Korea for more than half a century, from 1949 to 2005. During a major epidemic in 1949, there were 5616 cases and 2729 deaths reported, with levels persisting near epidemic levels of 1000 cases annually thereafter until 1969. Following the introduction and government mandated mass immunization in 1971, JE decreased dramatically. Since 1984, 0–6 cases of JE have been reported each year. However, continued evidence of mosquitoes positive for JEV indicates that JE continues to be a civilian and military health threat to immunocompromised persons in Korea, as well as non‐immune US soldiers, civilians and their family members.     

Detection of Japanese Encephalitis Virus Genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea

Japanese encephalitis virus (JEV) causes significant viral encephalitis and is distributed throughout the Asian countries. The virus is known to be transmitted by Culex tritaeniorhynchus, which mainly breeds in rice paddies in Korea. In this study, we investigated the presence of other mosquito species that can transmit JEV as a second or regional vector. We selected five cities where patients have experienced JE in the last 5 years as mosquito-collecting locations and subdivided them into four collection sites according to the mosquito habitats (cowshed, downtown area, forest, and swamp). Mosquitoes were caught using the BG-Sentinel trap, CDC black-light trap, Fay-Prince trap, and Gravid trap. A total of 993 pools from 22,774 mosquitoes were prepared according to their species, collection date, and site. We performed a SYBR Green 1-based real-time RT-PCR assay to detect JEV from the mosquito pools. A total of six JEV-positive pools were detected from Culex orientalis and Culex pipiens caught in the Gangwon-do and Gyeonngi-do provinces. All the detected JEVs were revealed as genotype V by phylogenetic analysis of the envelope gene. Our findings confirm that a new genotype of JEV was introduced in Korea and suggest that two mosquito species may play a role in JEV transmission.     

The current burden of Japanese encephalitis and the estimated impacts of vaccination: Combining estimates of the spatial distribution and transmission intensity of a zoonotic pathogen

Japanese encephalitis virus (JEV) is a major cause of neurological disability in Asia and causes thousands of severe encephalitis cases and deaths each year. Although Japanese encephalitis (JE) is a WHO reportable disease, cases and deaths are significantly underreported and the true burden of the disease is not well understood in most endemic countries. Here, we first conducted a spatial analysis of the risk factors associated with JE to identify the areas suitable for sustained JEV transmission and the size of the population living in at-risk areas. We then estimated the force of infection (FOI) for JE-endemic countries from age-specific incidence data. Estimates of the susceptible population size and the current FOI were then used to estimate the JE burden from 2010 to 2019, as well as the impact of vaccination. Overall, 1,543.1 million (range: 1,292.6-2,019.9 million) people were estimated to live in areas suitable for endemic JEV transmission, which represents only 37.7% (range: 31.6-53.5%) of the over four billion people living in countries with endemic JEV transmission. Based on the baseline number of people at risk of infection, there were an estimated 56,847 (95% CI: 18,003-184,525) JE cases and 20,642 (95% CI: 2,252-77,204) deaths in 2019. Estimated incidence declined from 81,258 (95% CI: 25,437-273,640) cases and 29,520 (95% CI: 3,334-112,498) deaths in 2010, largely due to increases in vaccination coverage which have prevented an estimated 314,793 (95% CI: 94,566-1,049,645) cases and 114,946 (95% CI: 11,421-431,224) deaths over the past decade. India had the largest estimated JE burden in 2019, followed by Bangladesh and China. From 2010-2019, we estimate that vaccination had the largest absolute impact in China, with 204,734 (95% CI: 74,419-664,871) cases and 74,893 (95% CI: 8,989-286,239) deaths prevented, while Taiwan (91.2%) and Malaysia (80.1%) had the largest percent reductions in JE burden due to vaccination. Our estimates of the size of at-risk populations and current JE incidence highlight countries where increasing vaccination coverage could have the largest impact on reducing their JE burden.     

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus,which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis.Axl,a receptor tyrosine kinase from TAM family,plays crucial role in many inflammatory diseases.We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection,which we speculate is due to the anti-inflammatory effect of Axl during JE.Currently,the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet.In this study,by using Axl deficient and heterozygous control mice,we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death,extended infiltration of inflammatory cells,and enhanced production of pro-inflammatory cytokines,in comparison to control mice.Additionally,consistent with our previous report,Axl deficiency had no impact on the infection and target cell tropism of JEV in brain.Taken together,our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.     

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

Background

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets.

Methodology/Principal Findings

A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested.

Conclusions/Significance

Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.     

我国2009年新分离乙脑病毒全基因组序列特征研究

本研究对我国2009年新分离的两株乙脑病毒进行全基因组序列测定和分析,以了解病毒全基因组分子特征。通过RT-PCR和核苷酸序列测定方法获得病毒全基因组序列,采用ClustalX、DNASTAR、MEGA等生物学软件完成核苷酸序列及氨基酸序列分析和系统进化分析等。研究结果显示,新分离两株乙脑病毒YN0911和YN0967株基因组全长均为10 965个核苷酸,编码3 432个氨基酸。这2株乙脑病毒之间核苷酸同源性为98.7%,氨基酸同源性为99.8%。与国际乙脑病毒流行株相比,核苷酸同源性为83.5%～98.9%,氨基酸同源性为94.8%～99.7%。与乙脑病毒疫苗株SA14-14-2相比,在E蛋白有13个氨基酸差异位点,但都位于抗原关键位点之外。这2株病毒在3′UTR区域存在11nt缺失。基于C/PrM区段、E基因、全基因组系统进化分析结果均显示新分离2株乙脑病毒为G I乙脑病毒,并且和越南、四川、贵州、广西以往的分离株遗传进化关系较近。本研究提示我国新分离的2株乙脑病毒均为G I乙脑病毒,决定病毒毒力的关键氨基酸位点未见明显变化。     

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Landscape in Neuronal Cells during JEV Infection

Virologica Sinica - Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in endemic regions of Asia. The neurotropism of JEV and its high-efficiency replication in neurons are...     

Molecular Epidemiology of Japanese Encephalitis Virus in Mosquitoes in Taiwan during 2005–2012

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine''s efficacy.     

乙型脑炎病毒结构蛋白C＋pre M和E基因的克隆与序列分析

目的对乙型脑炎病毒（Japanese encephalitis virus,JEV）结构蛋白基因进行扩增、克隆和序列测定。方法根据GenBank中发表的乙脑病毒结构蛋白基因C＋pre M基因和E序列,设计合成两对引物,以JEV Nakayama-NIH株RNA为模板,反转录并扩增目的cDNA,然后与pGEM-T easy载体连接并转化大肠杆菌DH5α菌株,提取的重组质粒用电泳、PCR和酶切鉴定后并进行测序。将JEV Nakayama-NIH与GenBank中报道的多株JEV结构蛋白C＋pre M基因和E基因序列进行了比较。结果Nakayama-NIH与其他参考株C＋pre M和E基因的核苷酸同源性为99%,证实扩增克隆的是JEV的结构蛋白C＋pre M和E基因。结论对JEV分子生物学特征进行了分析,为JEV特异性核酸探针的研制奠定了基础。     

不同年份生产的乙型脑炎减毒活疫苗SA14-14-2基因稳定性研究

为了研究不同年份生产的乙型脑炎减毒活疫苗病毒E蛋白基因稳定性,从分子水平控制乙型脑炎减毒活疫苗质量,确保疫苗安全性,本研究分析了不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列及编码的氨基酸序列,并与该疫苗原始种子、主种子、工作种子、乙脑病毒强、弱毒株进行比较。结果显示不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列与其原始种子、主种子、工作种子和基因库中登录的乙脑病毒弱毒株SA14-14-2的相应序列完全一致,与乙脑病毒强毒株SA14的E蛋白氨基酸序列比较有9个位点氨基酸发生了改变。不同年份生产的乙脑活疫苗病毒E蛋白基因稳定性表明该疫苗质量稳定、安全。     

Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І

Background

Japanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype ? (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains.

Methods

The recombinant virus PRV TK^?/gE^?/PrM-E⁺ which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK^?/gE^?/LacZ⁺ digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK^?/gE^?/PrM-E⁺ was further evaluated in mouse model.

Results

A recombinant pseudorabies virus (PRV TK^?/gE^?/PrM-E⁺) was successfully constructed. Mice experiments showed that PRV TK^?/gE^?/PrM-E⁺ could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1?×?10⁷ PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK^?/gE^?/PrM-E⁺ vaccinated mice.

Conclusions

PRV TK^?/gE^?/PrM-E⁺ is a potential vaccine candidate against PRV and JEV GI infection in the future.

     

Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.     

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang,an Eastern Province of China

BackgroundSporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains.Conclusion/SignificancesJE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.