Genotype v Japanese encephalitis virus is emerging

Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.     

Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.     

Circulation of Japanese Encephalitis Virus in Pigs and Mosquito Vectors within Can Tho City,Vietnam

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing encephalitis in humans and reproductive disorder in pigs. JEV is present in large parts of Asia, where urbanization is high. Households within and outside Can Tho city, South Vietnam, were selected to monitor circulation of JEV. A nested RT-PCR was established to detect the presence of JEV in mosquitoes whereas sera from pigs belonging to households within the province were analyzed for the presence of antibodies to JEV. A total of 7885 mosquitoes were collected and divided into 352 pools whereof seven were JEV-positive, six of which were collected within the city. Fragments from four pools clustered with JEV genotype III and three with genotype I. Of the 43 pigs sampled inside the city 100% had JEV antibodies. Our study demonstrates exposure to JEV in pigs, and co-circulation of JEV genotype I and III in mosquitoes within an urban environment in South Vietnam. Thus, although JEV has mainly been considered a rural disease, the potential for transmission in urban areas cannot be ignored.     

乙型脑炎疫苗研究进展

乙型脑炎（乙脑）的流行是世界性的公共卫生问题之一,其流行范围正逐步扩大。目前对于乙脑尚无特效治疗手段,因此预防乙脑的发生尤为重要。随着对日本脑炎病毒研究的深入,利用基因工程技术研制新型候选疫苗已成为预防乙脑新的发展方向。我们简要综述了乙型脑炎病毒的基因组结构及其蛋白功能,以及国内外乙型脑炎疫苗研发的新进展。     

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.     

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。     

Seroprevalence of Japanese encephalitis virus infection in captive Japanese macaques (Macaca fuscata)

Japanese encephalitis virus (JEV), which is transmitted by mosquitoes, infects many animal species and causes serious acute encephalitis in humans and horses. In this study, a serosurvey of JEV in Japanese macaques (Macaca fuscata) reared in Aichi Prefecture was conducted using purified JEV as an antigen for ELISA. The results revealed that 146 of 332 monkeys (44 %) were seropositive for JEV. In addition, 35 of 131 monkeys (27 %) born in the facility were seropositive, and the annual infection rate in the facility was estimated as 13 %. Our results provide evidence of the frequent exposure of many Japanese macaques to JEV, suggesting that there is a risk of JEV transmission to humans by mosquitoes.     

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group. JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis. H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV. Virus replication was found to be inhibited in the brains of animals that were adoptively transferred with JEV specific CTL as revealed by immunohistological staining as well as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.     

Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8?M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124?mg of highly purified and biologically active EDIII protein was obtained from 100?g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.     

Japanese encephalitis virus: from genome to infectome

Japanese encephalitis virus (JEV) is an arbovirus belonging to the family Flaviviridae. It is maintained in a zoonotic cycle involving pigs, ardeid birds and Culex species of mosquitoes. Humans are accidental/dead end hosts of JEV infection because they cannot sustain high viral titers. Factors affecting the clinical manifestations and pathogenesis of JEV infection are not well understood. Though, vaccines are currently available against JEV, it has to be further improved. Here we review the literature on the JEV life cycle, pathogenesis and host immune responses to JEV infection.     

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.     

The Chemokine Receptor CCR5, a Therapeutic Target for HIV/AIDS Antagonists,Is Critical for Recovery in a Mouse Model of Japanese Encephalitis

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 NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL

Virologica Sinica - Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a...     

Broad-spectrum antiviral activity of RNA interference against four genotypes of Japanese encephalitis virus based on single microRNA polycistrons

Japanese encephalitis virus (JEV), a neurotropic mosquito-borne flavivirus, causes acute viral encephalitis and neurologic disease with a high fatality rate in humans and a range of animals. Small interfering RNA (siRNA) is a powerful antiviral agent able to inhibit JEV replication. However, the high rate of genetic variability between JEV strains (of four confirmed genotypes, genotypes I, II, III and IV) hampers the broad-spectrum application of siRNAs, and mutations within the targeted sequences could facilitate JEV escape from RNA interference (RNAi)-mediated antiviral therapy. To improve the broad-spectrum application of siRNAs and prevent the generation of escape mutants, multiple siRNAs targeting conserved viral sequences need to be combined. In this study, using a siRNA expression vector based on the miR-155 backbone and promoted by RNA polymerase II, we initially identified nine siRNAs targeting highly conserved regions of seven JEV genes among strains of the four genotypes of JEV to effectively block the replication of the JEV vaccine strain SA14-14-2. Then, we constructed single microRNA-like polycistrons to simultaneously express these effective siRNAs under a single RNA polymerase II promoter. Finally, these single siRNAs or multiple siRNAs from the microRNA-like polycistrons showed effective anti-virus activity in genotype I and genotype III JEV wild type strains, which are the predominant genotypes of JEV in mainland China. The anti-JEV effect of these microRNA-like polycistrons was also predicted in other genotypes of JEV (genotypes II and IV), The inhibitory efficacy indicated that siRNAs×9 could theoretically inhibit the replication of JEV genotypes II and IV.     

Autophagy is involved in the early step of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry-LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.     

Endothelial Japanese encephalitis virus infection enhances migration and adhesion of leukocytes to brain microvascular endothelia via MEK‐dependent expression of ICAM1 and the CINC and RANTES chemokines

Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis‐associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV‐infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule‐1, cytokine‐induced neutrophil chemoattractant‐1, and regulated‐upon‐activation normal T‐cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal‐regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up‐regulating cytosolic phospholipase A₂, NF‐κB, and cAMP response element‐binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier‐disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.     

Impaired Japanese encephalitis virus replication in p62/SQSTM1 deficient mouse embryonic fibroblasts

The role of the autophagy adaptor protein p62/SQSTM1 in Japanese encephalitis virus (JEV) replication in mouse embryonic fibroblasts (MEFs) was investigated. Amounts of JEV RNA and E protein were significantly smaller in p62‐deficient cells than wild‐type cells at 24 hr post‐infection (p.i.). JEV RNA quantitation and viral plaque assays showed significant reductions in viral titers in p62‐deficient cell culture fluid. Our results indicate that JEV replication is impaired in p62‐deficient MEFs, suggesting that p62 positively regulates JEV replication in host cells.     

Estimating the Burden of Japanese Encephalitis Virus and Other Encephalitides in Countries of the Mekong Region

Diverse aetiologies of viral and bacterial encephalitis are widely recognized as significant yet neglected public health issues in the Mekong region. A robust analysis of the corresponding health burden is lacking. We retrieved 75 articles on encephalitis in the region published in English or in French from 1965 through 2011. Review of available data demonstrated that they are sparse and often derived from hospital-based studies with significant recruitment bias. Almost half (35 of 75) of articles were on Japanese encephalitis virus (JEV) alone or associated with dengue. In the Western Pacific region the WHO reported 30,000–50,000 annual JEV cases (15,000 deaths) between 1966 and 1996 and 4,633 cases (200 deaths) in 2008, a decline likely related to the introduction of JEV vaccination in China, Vietnam, or Thailand since the 1980s. Data on dengue, scrub typhus and rabies encephalitis, among other aetiologies, are also reviewed and discussed.Countries of the Mekong region are undergoing profound demographic, economic and ecological change. As the epidemiological aspects of Japanese encephalitis (JE) are transformed by vaccination in some countries, highly integrated expert collaborative research and objective data are needed to identify and prioritize the human health, animal health and economic burden due to JE and other pathogens associated with encephalitides.     

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.     

Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE)

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.