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Neurotropic viruses, characterized by their capacity to invade the central nervous system, present a considerable challenge to public health and are responsible for a diverse range of neurological disorders. This group includes a diverse array of viruses, such as herpes simplex virus, varicella zoster virus, poliovirus, enterovirus and Japanese encephalitis virus, among others. Some of these viruses exhibit high neuroinvasiveness and neurovirulence, while others demonstrate weaker neuroinvasive and neurovirulent properties. The clinical manifestations of infections caused by neurotropic viruses can vary significantly, ranging from mild symptoms to severe life-threatening conditions. Extracellular vesicles (EVs) have garnered considerable attention due to their pivotal role in intracellular communication, which modulates the biological activity of target cells via the transport of biomolecules in both health and disease. Investigating EVs in the context of virus infection is crucial for elucidating their potential role contribution to viral pathogenesis. This is because EVs derived from virus-infected cells frequently transfer viral components to uninfected cells. Importantly, EVs released by virus-infected cells have the capacity to traverse the blood-brain barrier (BBB), thereby impacting neuronal activity and inducing neuroinflammation. In this review, we explore the roles of EVs during neurotropic virus infections in either enhancing or inhibiting viral pathogenesis. We will delve into our current comprehension of the molecular mechanisms that underpin these roles, the potential implications for the infected host, and the prospective diagnostic applications that could arise from this understanding., Competing Interests: Declaration of competing interest The authors declare that they have no any conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals., (© 2024. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

Most wild strains of Japanese encephalitis virus (JEV) produce NS1' protein, which plays an important role in viral infection and immune escape. The G66A nucleotide mutation in NS2A gene of the wild strain SA14 prevented the ribosomal frameshift that prevented the production of NS1' protein, thus reduced the virulence. In this study, the 66th nucleotide of the NS2A gene of SA14 was mutated into A, U or C, respectively. Both the G66U and G66C mutations cause the E22D mutation of the NS2A protein. Subsequently, the expression of NS1' protein, plaque size, replication ability, and virulence to mice of the three mutant strains were examined. The results showed that the three mutant viruses could not express NS1' protein, and their proliferation ability in nerve cells and virulence to mice were significantly reduced. In addition, the SA14(G66C) was less virulent than the other two mutated viruses. Our results indicate that only when G is the 66th nucleotide of NS2A, the JEV can produce NS1' protein, which affects the virulence., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Japanese encephalitis (JE) is a mosquito-borne disease that causes neuronal damage and inflammation of microglia, and in severe cases, it can be fatal. JE infection can resist cellular immune responses and survive in host cells. Japanese encephalitis virus (JEV) infects macrophages and peripheral blood lymphocytes. In addition to regulating biological signaling pathways, microRNAs in cells also influence virus-host interactions. Under certain circumstances, viruses can change microRNA production. These changes affect the replication and spread of the virus. Host miRNAs can contain viral pathogenicity by downregulating the antiviral immune response pathways. Simultaneous profiling of miRNA and messenger RNA (mRNA) could help us detect pathogenic factors, and dual RNA detection is possible. This work highlights important miRNAs involved in human JE infection. In this study, we have shown the important miRNAs that play significant roles in JEV infection. We found that during JEV infection, miRNA-155, miRNA-29b, miRNA-15b, miRNA-146a, miRNA-125b-5p, miRNA-30la, miRNA-19b-3p, and miRNA-124, cause upregulation of human genes whereas miRNA-432, miRNA-370, miRNA- 33a-5p, and miRNA-466d-3p are responsible for downregulation of human genes respectively. Further, these miRNAs are also responsible for the inflammatory effects. Although several other miRNAs critical to the JEV life cycle are yet unknown, there is currently no evidence for the role of miRNAs in persistence., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Importance: Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1 . RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival., Competing Interests: The authors declare no conflict of interest.

Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus Flavivirus of the family Flaviviridae and has caused huge threat to public health. It is still obscure regarding the functions of stem-loop (SL) and dumbbell (DB) domains of JEV 3' UTR in viral replication and virulence. In the current study, using the infectious clone of JEV SA14 strain as a backbone, we constructed a series of deletion mutants of 3' UTR to investigate their effects on virus replication. The results showed that partial deletions within SL or DB domain had no apparent effects on virus replication in both mammalian (BHK-21) and mosquito (C6/36) cells, suggesting that they were not involved in viral host-specific replication. However, the entire SL domain deletion (ΔVR) significantly reduced virus replication in both cell lines, indicating the important role of the complete SL domain in virus replication. The revertant of ΔVR mutant virus was obtained by serial passage in BHK-21 cells that acquired a duplication of DB domain (DB-dup) in the 3' UTR, which greatly restored virus replication as well as the capability to produce the subgenomic flavivirus RNAs (sfRNAs). Interestingly, the DB-dup mutant virus was highly attenuated in C57BL/6 mice despite replicating similar to WT JEV. These findings demonstrate the significant roles of the duplicated structures in 3' UTR in JEV replication and provide a novel strategy for the design of live-attenuated vaccines.

Japanese encephalitis (JE), an acute encephalitis syndrome disease caused by infection with JE virus (JEV), is an important mosquito borne disease in developing countries. The clinical outcomes of JEV infection show inter individual differences. Only in a minor percent of the infected subjects, the disease progresses into acute encephalitis syndrome. Single nucleotide polymorphisms in the host immune response related genes are known to affect susceptibility to JE. In the present study, 238 JE cases and 405 healthy controls (HCs) without any known history of encephalitis were investigated for SNPs in the CD209 MX1, TLR3, MMP9, TNFA and IFNG genes which are important in the immune response against JEV by PCR based methods. The results revealed higher frequencies of heterozygous genotypes of CD209 rs4804803, MMP9 rs17576, TNFA rs1800629 and IFNG rs2430561 in JE cases compared to HCs. These SNPs were associated with JE in an over-dominant genetic model (Odds ratio with 95% CI 1.51 (1.09-2.10) for CD209 rs4804803, 1.52 (1.09-2.11) for MMP9 rs17576, and 1.55 (1.12-2.15) for IFNG rs2430561). The association of G/A genotype of TNFA rs1800629 with JE was confirmed in a larger sample size. The results suggest the association of CD209 rs4804803, MMP9 rs17576, IFNG rs2430561 and TNFA rs1800629 polymorphisms with susceptibility to JE., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in many regions of Asia. Cytokines, including pro-inflammatory and anti-inflammatory are key regulators playing a detrimental role in the host response to JE infection, pathogenesis and disease outcome. Evidently, the host's cytokine response is genetically determined, representing the complexity of interindividual differences regarding immune response to viral infection. The current study assesses the association of single nucleotide polymorphisms of classical interleukin IL-1β and IL-10 with JEV susceptibility and disease severity in north Indian population., Methods: We performed a case-control study using 85 JE patients and 85 healthy controls. Polymorphisms in the IL-1β (-511 C/T) and IL-10 (-1082 A/G) genes were genotyped using PCR-RFLP. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed in percentage., Results: The mRNA level of IL-1β and IL-10 were found significantly increased in JE patients. In severe JE patients, IL-1β mRNA level was significantly higher with heterozygous (C/T) and homozygous (C/C) genotype compared to wild (T/T) genotype and mRNA level of IL-10 was higher in heterozygous genotype (A/G) compared to wild genotype (A/A). The C/T and C/C genotypes of IL-1β were significantly associated with higher risk of JE infection (p < 0.05, OR = 7.25 and 4.40) whereas, the A/G genotype of IL-10 was associated with a reduced risk of JEV infection (p < 0.05, OR = 0.30). The C allele of IL-1β was associated with fever and neck stiffness (p < 0.05) and CT genotype was associated with disease severity and worse outcomes in JE patients. Along with this, IL-10 polymorphism was found associated with fever, and AG genotype was found to be associated with worse disease outcomes such as neurological sequelae (p < 0.05)., Conclusion: Mutant allele and genotype at IL-1β (-511 C/T) and IL-10 (-1082 A/G) gene polymorphism show increased expression of IL-1β and IL-10 in JE patients which contribute to disease severity as well as adverse outcomes of disease. Overall this is the first report from northern India, which shows the association of IL-1β and IL-10 polymorphisms with JEV infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Thousands of human deaths occur annually due to Japanese encephalitis (JE), caused by Japanese encephalitis virus. During the virus infection of the central nervous system, reactive gliosis, uncontrolled inflammatory response, and neuronal cell death are considered as the characteristic features of JE. To date, no specific treatment has been approved to overcome JE, indicating a need for the development of novel therapies. In this article, we focused on basic biological mechanisms in glial (microglia and astrocytes) and neuronal cells that contribute to the onset of neuroinflammation and neuronal cell damage during Japanese encephalitis virus infection. We also provided comprehensive knowledge about anti-JE therapies tested in clinical or pre-clinical settings, and discussed recent therapeutic strategies that could be employed for JE treatment. The improved understanding of JE pathogenesis might lay a foundation for the development of novel therapies to halt JE. Abbreviations AKT: a serine/threonine-specific protein kinase; AP1: activator protein 1; ASC: apoptosis-associated speck-like protein containing a CARD; ASK1: apoptosis signal-regulated kinase 1; ATF3/4/6: activating transcription factor 3/4/6; ATG5/7: autophagy-related 5/7; BBB: blood-brain barrier; Bcl-3/6: B-cell lymphoma 3/6 protein; CCL: C-C motif chemokine ligand; CCR2: C-C motif chemokine receptor 2; CHOP: C/EBP homologous protein; circRNA: circular RNA; CNS: central nervous system; CXCL: C-X-C motif chemokine ligand; dsRNA: double-stranded RNA; EDEM1: endoplasmic reticulum degradation enhancer mannosidase alpha-like 1; eIF2-ɑ: eukaryotic initiation factor 2 alpha; ER: endoplasmic reticulum; ERK: extracellular signal-regulated kinase; GRP78: 78-kDa glucose-regulated protein; ICAM: intercellular adhesion molecule; IFN: interferon; IL: interleukin; iNOS: inducible nitric oxide synthase; IRAK1/2: interleukin-1 receptor-associated kinase 1/2; IRE-1: inositol-requiring enzyme 1; IRF: interferon regulatory factor; ISG15: interferon-stimulated gene 15; JE: Japanese encephalitis; JEV: Japanese encephalitis virus; JNK: c-Jun N-terminal kinase; LAMP2: lysosome-associated membrane protein type 2; LC3-I/II: microtubule-associated protein 1 light chain 3-I/II; lncRNA: long non-coding RNA; MAPK: mitogen-activated protein kinase; miR/miRNA: microRNA; MK2: mitogen-activated protein kinase-activated protein kinase 2; MKK4: mitogen-activated protein kinase kinase 4; MLKL: mixed-linage kinase domain-like protein; MMP: matrix metalloproteinase; MyD88: myeloid differentiation factor 88; Nedd4: neural precursor cell-expressed developmentally downregulated 4; NF-κB: nuclear factor kappa B; NKRF: nuclear factor kappa B repressing factor; NLRP3: NLR family pyrin domain containing 3; NMDAR: N-methyl-D-aspartate receptor; NO: nitric oxide; NS2B/3/4: JEV non-structural protein 2B/3/4; P: phosphorylation. p38: mitogen-activated protein kinase p38; PKA: protein kinase A; PAK4: p21-activated kinase 4; PDFGR: platelet-derived growth factor receptor; PERK: protein kinase R-like endoplasmic reticulum kinase; PI3K: phosphoinositide 3-kinase; PTEN: phosphatase and tensin homolog; Rab7: Ras-related GTPase 7; Raf: proto-oncogene tyrosine-protein kinase Raf; Ras: a GTPase; RIDD: regulated IRE-1-dependent decay; RIG-I: retinoic acid-inducible gene I; RIPK1/3: receptor-interacting protein kinase 1/3; RNF11/125: RING finger protein 11/125; ROS: reactive oxygen species; SHIP1: SH2-containing inositol 5' phosphatase 1; SOCS5: suppressor of cytokine signaling 5; Src: proto-oncogene tyrosine-protein kinase Src; ssRNA = single-stranded RNA; STAT: signal transducer and activator of transcription; TLR: toll-like receptor; TNFAIP3: tumor necrosis factor alpha-induced protein 3; TNFAR: tumor necrosis factor alpha receptor; TNF-α: tumor necrosis factor-alpha; TRAF6: tumor necrosis factor receptor-associated factor 6; TRIF: TIR-domain-containing adapter-inducing interferon-β; TRIM25: tripartite motif-containing 25; VCAM: vascular cell adhesion molecule; ZO-1: zonula occludens-1.

A mouse-adapted isolate of Japanese encephalitis virus (JEV), designated as JEV-S3, was generated by serially passaging the P20778 strain of the virus in 3- to 4-week-old C57BL/6 mice. Blood-brain barrier leakage was evident in JEV-S3-infected mice, in which viral antigens and RNA were consistently demonstrated in the brain, along with infiltration of activated immune cells, as evidenced by an increased CD45+CD11b+ cell population. Histopathology studies showed the presence of perivascular cuffing, haemorrhage and necrotic foci in the virus-infected brain, conforming to the pathological changes seen in the brain of JEV-infected patients. Mass spectrometry studies characterized the molecular events leading to brain inflammation in the infected mice. Notably, a significant induction of inflammatory cytokines, such as IFNγ, IL6, TNFα and TGFβ, was observed. Further, genome sequencing of the JEV-S3 isolate identified the mutations selected during the mouse passage of the virus. Overall, we present an in-depth characterization of a robust and reproducible mouse model of JEV infection. The JEV-S3 isolate will be a useful tool to screen antivirals and study virus pathogenesis in the adolescent mouse model., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Infection with Japanese encephalitis virus (JEV) induces high morbidity and mortality, including potentially permanent neurological sequelae. However, the mechanisms by which viruses cross the blood-brain barrier (BBB) and invade into the central nervous system (CNS) remain unclear. Here, we show that extracellular HMGB1 facilitates immune cell transmigration. Furthermore, the migration of immune cells into the CNS dramatically increases during JEV infection which may enhance viral clearance, but paradoxically expedite the onset of Japanese encephalitis (JE). In this study, brain microvascular endothelial cells (BMECs) were utilized for the detection of HMGB1 release, and leucocyte, adhesion, and the integrity of the BBB in vitro . Genetically modified JEV-expressing EGFP (EGFP-JEV) and the BBB model were established to trace JEV-infected immune cell transmigration, which mimics the process of viral neuroinfection. We find that JEV causes HMGB1 release from BMECs while increasing adhesion molecules. Recombinant HMGB1 enhances leukocyte-endothelium adhesion, facilitating JEV-infected monocyte transmigration across endothelia. Thus, JEV successfully utilizes infected monocytes to spread into the brain, expanding inside of the brain, and leading to the acceleration of JE onset, which was facilitated by HMGB1. HMGB1-promoted monocyte transmigration may represent the mechanism of JEV neuroinvasion, revealing potential therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zou, Zou, Xiong, Cui, Wang, Liu, Lou, Higazy, Zhang and Cui.)

Background: Culex (Culex) tritaeniorhynchus is an important vector of Japanese encephalitis virus (JEV) affecting feral pigs, native mammals and humans. The mosquito species is widely distributed throughout Southeast Asia, Africa and Europe, and thought to be absent in Australia., Methods: In February and May, 2020 the Medical Entomology unit of the Northern Territory (NT) Top End Health Service collected Cx. tritaeniorhynchus female specimens (n = 19) from the Darwin and Katherine regions. Specimens were preliminarily identified morphologically as the Vishnui subgroup in subgenus Culex. Molecular identification was performed using cytochrome c oxidase subunit 1 (COI) barcoding, including sequence percentage identity using BLAST and tree-based identification using maximum likelihood analysis in the IQ-TREE software package. Once identified using COI, specimens were reanalysed for diagnostic morphological characters to inform a new taxonomic key to related species from the NT., Results: Sequence percentage analysis of COI revealed that specimens from the NT shared 99.7% nucleotide identity to a haplotype of Cx. tritaeniorhynchus from Dili, Timor-Leste. The phylogenetic analysis showed that the NT specimens formed a monophyletic clade with other Cx. tritaeniorhynchus from Southeast Asia and the Middle East. We provide COI barcodes for most NT species from the Vishnui subgroup to aid future identifications, including the first genetic sequences for Culex (Culex) crinicauda and the undescribed species Culex (Culex) sp. No. 32 of Marks. Useful diagnostic morphological characters were identified and are presented in a taxonomic key to adult females to separate Cx. tritaeniorhynchus from other members of the Vishnui subgroup from the NT., Conclusions: We report the detection of Cx. tritaeniorhynchus in Australia from the Darwin and Katherine regions of the NT. The vector is likely to be already established in northern Australia, given the wide geographical spread throughout the Top End of the NT. The establishment of Cx. tritaeniorhynchus in Australia is a concern to health officials as the species is an important vector of JEV and is now the sixth species from the subgenus Culex capable of vectoring JEV in Australia. We suggest that the species must now be continuously monitored during routine mosquito surveillance programmes to determine its current geographical spread and prevent the potential transmission of exotic JEV throughout Australia., (© 2021. The Author(s).)

West Nile virus disease (WND) is an arthropod-borne zoonosis responsible for nonspecific fever or severe encephalitis. The pathogen is West Nile virus belonging to the genus Flavivirus, family Flaviviridae . Every year, thousands of cases were reported, which poses significant public health risk. Here, we constructed a West Nile virus chimera, ChiVax-WN01, by replacing the prMΔE gene of JEV SA14-14-2 with that of the West Nile virus NY99. The ChiVax-WN01 chimera showed clear, different characters compared with that of JEV SA14-14-2 and WNV NY99 strain. An animal study indicated that the ChiVax-WN01 chimera presented moderate safety and immunogenicity for 4-week female BALB/c mice.

Background: The crucial role of type I interferon (IFN-I, IFN-α/β) is well known to control central nervous system (CNS) neuroinflammation caused by neurotrophic flaviviruses such as Japanese encephalitis virus (JEV) and West Nile virus. However, an in-depth analysis of IFN-I signal-dependent cellular factors that govern CNS-restricted tropism in JEV infection in vivo remains to be elucidated., Methods: Viral dissemination, tissue tropism, and cytokine production were examined in IFN-I signal-competent and -incompetent mice after JEV inoculation in tissues distal from the CNS such as the footpad. Bone marrow (BM) chimeric models were used for defining hematopoietic and tissue-resident cells in viral dissemination and tissue tropism., Results: The paradoxical and interesting finding was that IFN-I signaling was essentially required for CNS neuroinflammation following JEV inoculation in distal footpad tissue. IFN-I signal-competent mice died after a prolonged neurological illness, but IFN-I signal-incompetent mice all succumbed without neurological signs. Rather, IFN-I signal-incompetent mice developed hemorrhage-like disease as evidenced by thrombocytopenia, functional injury of the liver and kidney, increased vascular leakage, and excessive cytokine production. This hemorrhage-like disease was closely associated with quick viral dissemination and impaired IFN-I innate responses before invasion of JEV into the CNS. Using bone marrow (BM) chimeric models, we found that intrinsic IFN-I signaling in tissue-resident cells in peripheral organs played a major role in inducing the hemorrhage-like disease because IFN-I signal-incompetent recipients of BM cells from IFN-I signal-competent mice showed enhanced viral dissemination, uncontrolled cytokine production, and increased vascular leakage. IFN-I signal-deficient hepatocytes and enterocytes were permissive to JEV replication with impaired induction of antiviral IFN-stimulated genes, and neuron cells derived from both IFN-I signal-competent and -incompetent mice were vulnerable to JEV replication. Finally, circulating CD11b + Ly-6C + monocytes infiltrated into the distal tissues inoculated by JEV participated in quick viral dissemination to peripheral organs of IFN-I signal-incompetent mice at an early stage., Conclusion: An IFN-I signal-dependent model is proposed to demonstrate how CD11b + Ly-6C + monocytes are involved in restricting the tissue tropism of JEV to the CNS.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA's involvement in flavivirus persistent infection. Here, we used an miRNA array analysis of Japanese encephalitis virus (JEV)-infected cells to search for persistent infection-associated miRNAs in comparison to acute infection. Among all differentially expressed miRNAs, the miR-125b-5p is the most significantly increased one. The high level of miR-125b-5p in persistently JEV-infected cells was confirmed by Northern analysis and real-time quantitative polymerase chain reaction. As soon as the cells established a persistent infection, a significantly high expression of miR-125b-5p was readily observed. Transfecting excess quantities of a miR-125b-5p mimic into acutely infected cells reduced genome replication and virus titers. Host targets of miR125b-5p were analyzed by target prediction algorithms, and six candidates were confirmed by a dual-luciferase reporter assay. These genes were upregulated in the acutely infected cells and sharply declined in the persistently infected cells. The transfection of the miR125b-5p mimic reduced the expression levels of Stat3, Map2k7, and Triap1. Our studies indicated that miR-125b-5p targets both viral and host sequences, suggesting its role in coordinating viral replication and host antiviral responses. This is the first report to characterize the potential roles of miR-125b-5p in persistent JEV infections.

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death and disability whose primary vector is the Culex mosquito. Viral infection induces a series of antimicrobial peptide responses in mosquitoes, and the effector defensin enhances JEV replication in mosquitoes. However, the underlying mechanisms by which defensin enhances JEV are not fully understood. Here, we found that mosquito defensin could downregulate the antiviral protein HSC70B and enhance virus infection in mosquitoes. The cell-surface protein HSC70B was significantly downregulated by JEV infection and defensin treatment. Low levels of HSC70B were beneficial to JEV infection in mosquitoes. Taken together, these findings show that defensin and HSC70B axis facilitates JEV infection in the mosquito., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although Japanese encephalitis virus (JEV) infection is an important cause of acute febrile illness in Lao PDR (Laos), patient outcome has not been evaluated. We prospectively followed up 123 JEV-infected patients (70 children < 15 years and 53 adults ≥ 15 years) admitted at Mahosot Hospital, Vientiane, from 2003 to 2013. Japanese encephalitis virus infection was diagnosed by the detection of anti-JEV IgM in cerebrospinal fluid and/or IgM seroconversion. Neurological sequelae were assessed using the Liverpool Outcome Score (LOS), total (maximum score = 75), and final (maximum score = 5). The median (interquartile range [IQR]) age of the patients was 12.0 (7.5-18.8) years, and 57% were male. The median (IQR) duration of patients' follow-up was 4.5 (3.2-7.3) years. Of all patients, 10/123 (8.1%) died during hospitalization, and 13/123 (10.6%) died at home after discharge, giving a mortality of 18.7% (23/123) (33 [26.8%] patients were lost to follow-up). The frequency of neurological sequelae at the last follow-up was 61.2% (48.4% in adults and 69.4% in children, P = 0.135). The proportion of patients with severe and moderate functional impairment at the last follow-up was significantly higher in children (25%) than in adults (6.5%), P = 0.042. Half of the patients who were still alive at the last follow-up (67) and for whom LOS data were available (22) had improvements in their total and final LOS between discharge and the last follow-up. The total and final LOS at discharge were not significantly different between children and adults, but total LOS at the last follow-up was significantly higher in adults than in children (median [IQR]: 74.5 [73-75] versus 73.0 [73-75], P = 0.019).

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

Japanese encephalitis virus (JEV) is one of the major causes of viral encephalitis all around the globe. Approximately 3 billion people in endemic areas are at risk of Japanese encephalitis. To develop a wholistic understanding of the viral proteome, it is important to investigate both its ordered and disordered proteins. However, the functional and structural significance of disordered regions in the JEV proteome has not been systematically investigated as of yet. To fill this gap, we used here a set of bioinformatics tools to analyze the JEV proteome for the predisposition of its proteins for intrinsic disorder and for the presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs). We also analyzed all JEV proteins for the presence of the probable nucleic acid-binding (DNA and RNA) sites. The results of these computational studies are experimentally validated using JEV capsid protein as an illustrative example. In agreement with bioinformatic analysis, we found that the N-terminal region of the JEV capsid (residues 1-30) is intrinsically disordered. We showed that this region is characterized by the temperature response typical for highly disordered proteins. Furthermore, we have experimentally shown that this disordered N-terminal domain of a capsid protein has a noticeable 'gain-of-structure' potential. In addition, using DOPS liposomes, we demonstrated the presence of pronounced membrane-mediated conformational changes in the N-terminal region of JEV capsid. In our view, this disorder-centric analysis would be helpful for a better understanding of the JEV pathogenesis., (© 2020 Federation of European Biochemical Societies.)

Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation, neurobehavioral function and neuronal death using a mouse model of JEV. Markers for autophagy (LC3-II/I, SQSTM1/P62, phos-Akt, phos-AMPK), and neuronal death (cleaved caspase 12, H2Ax, polyubiquitin) were investigated by western blot at 1, 3 and 7 days post inoculation. Cathepsin D was measured in cerebral cotex of JEV infected mice spectrophotometrically. Microglia activation and pro-inflammatory cytokines (IL1β, TNF-α, IFNγ, IL6) were measured by immunohistochemistry, western blot and qPCR analysis. In order to determine the neuroinflammatory changes and autophagy mediated neuronal cell death, BV2-microglia and N2a-neuronal cells were used. Autophagy activation marker LC3-II/I and its substrate SQSTM1/P62 were significantly increased while cathepsin D activity was decreased on day 7 post inoculation in cerebral cortex. Microglia in cortex were activated and showed higher expression of proinflammatory mRNA of IL1β, TNF-α, IFNγ and IL6, with increased DNA damage (H2AX) and neuronal cell death pathways in hippocampus and neurobehavioral dysfunction. Similar observations on JEV infection mediated autophagy flux inhibition and neuronal cell death was found in N2a neuronal cell. Collectively, our study provides evidence on the role of autophagy regulation, microglial activation and neurodegeneration following JEV infection.

Background: Japanese encephalitis virus (JEV) is one of the common causes of acute encephalitis in humans. Japanese encephalitis is characterized by the uncontrolled release of inflammatory cytokines, which ultimately results in neuronal cell damage. In recent years, with the advancement of high-throughput sequencing technology, studies have shown that circRNAs, by competing with endogenous miRNAs, play a vital role in the pathology of CNS diseases. However, it is unknown whether circRNAs participate in JEV-induced neuroinflammation., Results: By employing Illumina RNA-sequencing, we identified 180 circRNAs and 58 miRNAs that showed significant differential expression in JEV-infected mice brain tissues. The functional enrichment analyses revealed that these differentially regulated circRNAs were predominantly related to neurotransmission, histone modifications, transcription misregulation, and inflammation-associated calcium signaling pathway. Our established competing endogenous RNA (ceRNA) interaction network suggested the correlation of several circRNAs, miRNAs, and mRNAs in regulating the inflammatory response during JEV infection. Among the predicted interactions, the correlation between circ&#95;0000220, miR-326-3p, and BCL3/MK2/TRIM25 mRNAs was experimentally validated by knockdown or overexpression of the non-coding RNA entities in cultured mouse microglia. The knockdown of circ&#95;0000220 or overexpression of miR-326-3p caused a lower production of JEV-induced inflammatory cytokines., Conclusions: Conclusively, our study provides new insights into the host response to JEV infection and proposes the circRNA-targeting therapeutic interventions to rein in Japanese encephalitis.

Background: Japanese encephalitis (JE) occurs in fewer than 1% of JE virus (JEV) infections, often with catastrophic sequelae including death and neuropsychiatric disability. JEV transmission in Pakistan was documented in 1980s and 1990s, but recent evidence is lacking. Our objective was to investigate JEV as a cause of acute encephalitis in Pakistan., Methods: Persons aged ≥1 month with possible JE admitted to two acute care hospitals in Karachi, Pakistan from April 2015 to January 2018 were enrolled. Cerebrospinal fluid (CSF) or serum samples were tested for JEV immunoglobulin M (IgM) using the InBios JE DetectTM assay. Positive or equivocal samples had confirmatory testing using plaque reduction neutralization tests., Results: Among 227 patients, testing was performed on CSF in 174 (77%) and on serum in 53 (23%) patients. Six of eight patient samples positive or equivocal for JEV IgM had sufficient volume for confirmatory testing. One patient had evidence of recent West Nile virus (WNV) neurologic infection based on CSF testing. One patient each had recent dengue virus (DENV) infection and WNV infection based on serum results. Recent flavivirus infections were identified in two persons, one each based on CSF and serum results. Specific flaviviruses could not be identified due to serologic cross-reactivity. For the sixth person, JEV neutralizing antibodies were confirmed in CSF but there was insufficient volume for further testing., Conclusions: Hospital-based JE surveillance in Karachi, Pakistan could not confirm or exclude local JEV transmission. Nonetheless, Pakistan remains at risk for JE due to presence of the mosquito vector, amplifying hosts, and rice irrigation. Laboratory surveillance for JE should continue among persons with acute encephalitis. However, in view of serological cross-reactivity, confirmatory testing of JE IgM positive samples at a reference laboratory is essential., Competing Interests: The authors have declared that no competing interests exist.

The phenotypic and genotypic characteristics of a live-attenuated genotype I (GI) strain (SD12-F120) of Japanese encephalitis virus (JEV) were compared with its virulent parental SD12 strain to gain an insight into the genetic changes acquired during the attenuation process. SD12-F120 formed smaller plaque on BHK-21 cells and showed reduced replication in mouse brains compared with SD12. Mice inoculated with SD12-F120 via either intraperitoneal or intracerebral route showed no clinical symptoms, indicating a highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. SD12-F120 harbored 29 nucleotide variations compared with SD12, of which 20 were considered silent nucleotide mutations, while nine resulted in eight amino acid substitutions. Comparison of the amino acid variations of SD12-F120 vs SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the variations at E 138 and E 176 positions of E protein were identified in four and three pairs, respectively, while the remaining amino acid variations were almost unique to their respective strain pairs. These observations suggest that the genetic changes acquired during the attenuation process were likely to be strain-specific and that the mechanisms associated with JEV attenuation/virulence are complicated.

Japanese encephalitis (JE) poses a serious threat to the world's public health yet without a cure, the only way to prevent Japanese encephalitis virus (JEV) infection is vaccination. Live attenuated vaccine (SA14-14-2 strain) is the most widely used JE vaccine, and clinical data have confirmed its safety and effectiveness. Eight sitesassociated with virulence in the Envelope (E) protein are often the focus of quality control of JE vaccine. However, sequences retrieved from NCBI, as well as our previous results showed that the wild strain SA14 may harbor two different amino acids at amino acid residue 244 of the E glycoprotein (E244), and it may be related to virulence. In this study, we introduced a single mutation at nt1708 (G → A) in the full-length cDNA clone of SA14-14-2, replacing a Gly with Glu at amino acid residue 244 of the E glycoprotein, and successfully constructed the mutant virus (JEV E244). JEV E244 exhibited a similar plaque morphology and growth characteristics to JEV SA14-14-2 in cell culture. However, it had lethal neurovirulence in mice and could enter the brain following intraperitoneal inoculation. Moreover, the virulence of JEV E244 in the context of vaccine in mice is significantly different from that of the JEV E244 alone. These results suggested that E244 site should be included in the assessment of the genetic stability of the attenuated JE vaccine. The detection of minor mutations in vaccine population and influence on the safety of vaccine is discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide that is associated with high morbidity and mortality. Currently, there are no effective drugs approved for the treatment of JEV infection. Drug-repurposing screening is an alternative approach to discover potential antiviral agents. In this study, high-content screening (HCS) of a natural extracts library was performed, and two hit FDA-approved Na + /K + -ATPase inhibitors, ouabain and digoxin, were identified as having robust efficiency against JEV infection with the selectivity indexes over 1,000. The results indicated that ouabain and digoxin blocked the JEV infection at the replication stage by targeting the Na + /K + -ATPase. Furthermore, it was proven that ouabain significantly reduced the morbidity and mortality caused by JEV in a BALB/c mouse model. This work demonstrated that Na + /K + -ATPase could serve as the target of treatment of JEV infection, and ouabain has the potential to be developed as an effective anti-JEV drug., (Copyright © 2020 Guo et al.)

Background & Objectives: Mouse is a preferred animal model for studying pathogenesis of Japanese encephalitis virus (JEV) infections, and different routes of inoculation have been tried. Some neurotropic viruses can reach the brain following infection through ocular route. This study was undertaken to establish JEV-induced clinical disease in mouse model through conjunctival route and document the neuropathological effects., Methods: Ten two-week old Swiss albino mice were inoculated with 5 μl Vero cell cultured virus containing 10 4.7 TCID 50 JEV through conjunctival route. Clinical signs of mice were observed twice daily. After necropsy examination, different organs including eyes and olfactory bulbs were collected for histopathological examination, quantification of viral copy number and antigen by real-time TaqMan assay and immunohistochemistry, respectively., Results: Infected mice showed characteristic clinical signs of JE by 4 days post-infection (dpi). Histopathological lesions in brain included perivascular cuffing by mononuclear cells, focal gliosis, necrosis of neurons and neuronophagia and astrocytosis in the cerebrum, cerebellum and the brainstem. JEV viral load was highest in the brain followed by intestine, heart, liver, spleen, lung and kidney. JEV antigen was detected in the bipolar and ganglion cells of the retina and in the mitral cells and periglomerular cells of olfactory bulb and other parts of the brain., Interpretation & Conclusions: JEV infection in mice through conjunctival route produced characteristic clinical signs of the disease and neuropathological lesions. Demonstration of JEV antigen in association with neuropathological lesions in the central nervous system and neuronal cells of the eye showed that conjunctival route could be an effective alternate route for virus invasion into the brain. These findings have biosafety implications for researchers, veterinary practitioners and pig farmers., Competing Interests: None

Despite the existence of human vaccines, Japanese encephalitis (JE) remains the leading cause of human encephalitis in Asia. Pigs are described as the main amplifying host, but their role in JE epidemiology needs to be reassessed in order to identify and implement efficient control strategies, for both human and animal health. We aimed to provide a systematic review of publications linked to JE in swine, in terms of both individual and population characteristics of JE virus (JEV) infection and circulation, as well as observed epidemiological patterns. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement to select and analyze relevant articles from the Scopus database, 127 of which were included in the review. Pigs are central, but the implication of secondary hosts cannot be ruled out and should be further investigated. Although human vaccination cannot eradicate the virus, it is clearly the most important means of preventing human disease. However, a better understanding of the actual involvement of domestic pigs as well as other potential JEV hosts in different JEV epidemiological cycles and patterns could help to identify additional/complementary control measures, either by targeting pigs or not, and in some specific epidemiological contexts, contribute to reduce virus circulation and protect humans from JEV infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

RNA viruses are known to modulate host microRNA (miRNA) machinery for their own benefit. Japanese encephalitis virus (JEV), a neurotropic RNA virus, has been reported to manipulate several miRNAs in neurons or microglia. However, no report indicates a complete sketch of the miRNA profile of neural stem/progenitor cells (NSPCs), hence the focus of our current study. We used an miRNA array of 84 miRNAs in uninfected and JEV-infected human neuronal progenitor cells and primary neural precursor cells isolated from aborted fetuses. Severalfold downregulation of hsa-miR-9-5p, hsa-miR-22-3p, hsa-miR-124-3p, and hsa-miR-132-3p was found postinfection in both of the cell types compared to the uninfected cells. Subsequently, we screened for the target genes of these miRNAs and looked for the biological pathways that were significantly regulated by the genes. The target genes involved in two or more pathways were sorted out. Protein-protein interaction (PPI) networks of the miRNA target genes were formed based on their interaction patterns. A binary adjacency matrix for each gene network was prepared. Different modules or communities were identified in those networks by community detection algorithms. Mathematically, we identified the hub genes by analyzing their degree centrality and participation coefficient in the network. The hub genes were classified as either provincial ( P  < 0.4) or connector ( P  > 0.4) hubs. We validated the expression of hub genes in both cell line and primary cells through qRT-PCR after JEV infection and respective miR mimic transfection. Taken together, our findings highlight the importance of specific target gene networks of miRNAs affected by JEV infection in NSPCs. IMPORTANCE JEV damages the neural stem/progenitor cell population of the mammalian brain. However, JEV-induced alteration in the miRNA expression pattern of the cell population remains an open question, hence warranting our present study. In this study, we specifically address the downregulation of four miRNAs, and we prepared a protein-protein interaction network of miRNA target genes. We identified two types of hub genes in the PPI network, namely, connector hubs and provincial hubs. These two types of miRNA target hub genes critically influence the participation strength in the networks and thereby significantly impact up- and downregulation in several key biological pathways. Computational analysis of the PPI networks identifies key protein interactions and hubs in those modules, which opens up the possibility of precise identification and classification of host factors for viral infection in NSPCs., (Copyright © 2019 Mukherjee et al.)

Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis. IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis., (Copyright © 2019 American Society for Microbiology.)

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle., Competing Interests: The authors have declared that no competing interests exist.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Japanese encephalitis virus (JEV), a causative agent of severe viral encephalitis in humans, has a biological cycle fluctuating between transmission in mosquitoes and avian species and amplification in pigs. Contact transmission of JEV was recently shown in pigs in the absence of arthropod vectors. Here, we show JEV transmission between infected and contact mice and further demonstrate that JEV transmission occurs between animals via aerosols, as both viral RNA and infectious JEV were detected in direct contact- and aerosol-exposed contact animals. The results of this study change our understanding of JEV transmission in densely populated regions and may help to explain JEV outbreaks without the presence of arthropod vectors.

Background: Continuous vector pathogen surveillance is essential for preventing outbreaks of mosquito-borne diseases. Several mosquito species acting as vectors of Japanese encephalitis virus (JEV), dengue virus, Zika virus, malaria parasites and other pathogens are primary mosquito species in Shanghai, China. However, few surveys of human pathogenic arboviruses in mosquitoes in Shanghai have been reported in the last ten years. Therefore, in this study, we evaluated mosquito activity in Shanghai, China during 2016 and tested for the presence of alphaviruses, flaviviruses, orthobunyaviruses and several parasitic pathogens., Results: Five pooled samples were JEV-positive [4/255 pools of Culex tritaeniorhynchus and 1/256 pools of Cx. pipiens (s.l.)] based on analysis of the NS5 gene. Alphaviruses, orthobunyaviruses, Plasmodium and filariasis were not found in this study. Phylogenetic and molecular analyses revealed that the JEV strains belonged to genotype I. Moreover, newly detected Shanghai JEV strains were genetically close to previously isolated Shandong strains responsible for transmission during the 2013 Japanese encephalitis (JE) outbreak in Shandong Province, China but were more distantly related to other Shanghai strains detected in the early 2000s. The E proteins of the newly detected Shanghai JEV strains differed from that in the live attenuated vaccine SA14-14-2-derived strain at six amino residues: E130 (Ile→Val), E222 (Ala→Ser), E327 (Ser→Thr), E366 (Arg→Ser/Pro), E393 (Asn→Ser) and E433 (Val→Ile). However, no differences were observed in key amino acid sites related to antigenicity. Minimum JEV infection rates were 1.01 and 0.65 per 1000 Cx. tritaeniorhynchus and Cx. pipiens (s.l.), respectively., Conclusions: Five new Shanghai JEV genotype I strains, detected after a ten-year hiatus in local mosquito surveillance, were genetically close to strains involved in the 2013 Shandong JE outbreak. Because JEV is still circulating, vaccination in children should be extensively and continuously promoted. Moreover, JEV mosquito surveillance programmes should document the genotype variation, intensity and distribution of circulating viruses for use in the development and implementation of disease prevention and control strategies.

Japanese encephalitis is a zoonotic disease caused by the Japanese encephalitis virus (JEV). It is mainly epidemic in Asia with an estimated 69,000 cases occurring per year. However, no approved agents are available for the treatment of JEV infection, and existing vaccines cannot control various types of JEV strains. Drug repurposing is a new concept for finding new indication of existing drugs, and, recently, the concept has been used to discover new antiviral agents. Identifying host proteins involved in the progress of JEV infection and using these proteins as targets are the center of drug repurposing for JEV infection. In this study, based on the gene expression data of JEV infection and the phenome-wide association study (PheWAS) data, we identified 286 genes that participate in the progress of JEV infection using systems biology methods. The enrichment analysis of these genes suggested that the genes identified by our methods were predominantly related to viral infection pathways and immune response-related pathways. We found that bortezomib, which can target these genes, may have an effect on the treatment of JEV infection. Subsequently, we evaluated the antiviral activity of bortezomib using a JEV-infected mouse model. The results showed that bortezomib can lower JEV-induced lethality in mice, alleviate suffering in JEV-infected mice and reduce the damage in brains caused by JEV infection. This work provides an agent with new indication to treat JEV infection.

Autophagy is an evolutionarily conserved cellular process in which intracellular components are eliminated via lysosomal degradation to supply nutrients for organelle biogenesis and metabolic homeostasis. Flavivirus infections underlie multiple human diseases and thus exert an immense burden on public health worldwide. Mounting evidence indicates that host autophagy is subverted to modulate the life cycles of flaviviruses, such as hepatitis C virus, dengue virus, Japanese encephalitis virus, West Nile virus and Zika virus. The diverse interplay between autophagy and flavivirus infection not only regulates viral growth in host cells but also counteracts host stress responses induced by viral infection. In this review, we summarize the current knowledge on the role of autophagy in the flavivirus life cycle. We also discuss the impacts of virus-induced autophagy on the pathogeneses of flavivirus-associated diseases and the potential use of autophagy as a therapeutic target for curing flavivirus infections and related human diseases., Competing Interests: The author declares no conflict of interest.

Artificial microRNA (amiRNA)-mediated inhibition of viral replication has recently gained importance as a strategy for antiviral therapy. In this study, we evaluated the benefit of using the amiRNA vector against Japanese encephalitis virus (JEV). We designed three single amiRNA sequences against the consensus sequence of 3' untranslated region (3'UTR) of JEV and tested their efficacy against cell culture-grown JEV Vellore strain (P20778) in neuronal cells. The binding ability of three amiRNAs on 3'UTR region was tested in vitro in HEK293T cells using a JEV 3'UTR tagged with luciferase reporter vector. Transient transfection of amiRNAs was nontoxic to cells as evident from the MTT assay and caused minimal induction in interferon-stimulated gene expression. Furthermore, our result suggested that transient expression of two amiRNAs (amiRNA #1 and amiRNA #2) significantly reduced intracellular viral RNA and nonstructural 1 (NS1) protein, as well as diminished infectious viral particle release up to 95% in the culture supernatant as evident from viral plaque reduction assay. Overall, our results indicated that RNA interference based on amiRNAs targeting viral conserved regions at 3'UTR was a useful approach for improvements of nucleic acid inhibitors against JEV.

Japanese encephalitis virus (JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a 52-amino acid C-terminal extension of NS1, is generated with a -1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected (rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus (pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type I interferon (IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-β and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.

Japanese encephalitis (JE) is the largest worldwide cause of infectious encephalitis in humans and is caused by a mosquito-borne flavivirus. JE transmission cycle involves Culex mosquitoes, pigs and aquatic birds as principal vertebrate amplifying hosts. JE infection is responsible for reproductive disorder in pigs when occurring after sexual maturity. In tropical areas, JE is endemic and the majority of pigs get infected before the age of 6 months. However, in subtropical areas, pigs may be infected after sexual maturity and thus experience clinical signs, inducing economic loss. The study aimed at better characterizing the influence of seasonal temperature variations (through estimates of degree days, DD) on JE circulation in pigs in subtropical area and inferring on the potential impact on JE symptomatic infection in reproductive pigs. Six hundred and forty-one pig's sera sampled in northern Vietnam were analysed for Japanese encephalitis virus (JEV) by pan-flavivirus ELISA. A subset of 108 ELISA-positive samples, representative of each sampling occasion, were confirmed by JEV neutralization test compared with West Nile virus neutralization test. We modelled the seroprevalence of pigs according to a DD variable using a generalized additive model. We then predicted the age of infection in pigs according to their month of birth, using averaged temperature data over 10 years. The model predicts that only 80 percentage of pigs born between July and September will be protected against JEV when reaching sexual maturity contrary to the rest of the year when almost all pigs will seroconvert before sexual maturity. In subtropical area such as northern Vietnam, pigs could thus show symptomatic infection due to JE, and consequently reproductive disorders. Vaccination of future breeder pigs in epidemic areas could avoid the occurrence of JE-associated reproductive disorders., (© 2018 Blackwell Verlag GmbH.)

Japanese encephalitis virus (JEV) is a zoonotic flavivirus that is transmitted by mosquitoes and vertebrate-amplifying hosts, including birds. Domestic ducks are susceptible to JEV infection and develop various levels of viremia. We tested the pathogenicities of seven JEV strains in newly hatched domestic ducklings. All inoculated ducklings showed stunted growth. Two JEV strains caused notable mortalities of 12.7% and 31.7%, respectively, highlighting that some emerged JEV strains may thus be pathogenic in newly hatched domestic ducklings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Japanese encephalitis virus (JEV) is most important cause of viral encephalitis worldwide. The pathogenesis of this is probably attributed to the host genetic makeup. Intercellular adhesion molecule-1 (ICAM-1) and monocytes chemoattractant protein-1 (MCP-1) play a vital role in host defense mechanism against flavivirus causing encephalitis. We assessed the possible genetic association between ICAM-1 (K469E) and MCP-1-2518 A > G polymorphisms and Japanese Encephalitis in North Indian population., Methods: We studied ICAM-1(K469E) and MCP-1-2518 A > G polymorphisms with the help of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Expression of ICAM-1 and MCP-1 were determined at mRNA and protein levels in JE patients and healthy controls by real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA)., Results: Homozygous (E/E) genotype of ICAM-1 was associated with clinical severity (p = 0.015) and outcome (p = 0.04) of JE, whereas, heterozygous (A/G) genotype of MCP-1-2518 A > G was associated with outcome in JE patients (p = 0.01). Among severe cases of JE, a higher level of ICAM-1 was observed in patients with E allele (E/K + E/E) of ICAM-1 (K469E) than non-E allele (K/K). The level of MCP-1 was found significantly increased in JE patients with homozygous (G/G) genotype when compared to wild (A/A) genotype of MCP-1-2518 A > G (p = 0.03)., Conclusion: ICAM-1 (K469E) and MCP-1-2518 A > G polymorphisms lead to increased level of ICAM-1 and MCP-1 in Japanese Encephalitis which may be associated with severity as well as an adverse outcome of the disease. ICAM-1 (K469E) polymorphism may affect host susceptibility to Japanese encephalitis in North Indian population., (Copyright © 2018. Published by Elsevier Ltd.)

The Japanese encephalitis virus (JEV) envelope (E) protein, as one of mediators of virus entry into host cells, plays a critical role in determining virulence. The Glu-to-Lys mutation of residue 138 in E protein (E138) plays an important role in attenuating JEV vaccine strain SA14-14-2. However, it is not clear how E138 attenuates JEV. Here, we demonstrate that the Glu-to-Arg mutation of E138 also determines the attenuation of JEV strain 10S3. Likewise, for its parent strain (HEN0701), a virulence strain, the mutations of E138 are responsible for virulence alteration. Furthermore, we demonstrated that mutations of alkaline residues in E138 contributed to the attenuation of neurovirulence; in contrast, mutations of acidic residues enhanced the neurovirulence of the strains. Moreover, acidity in residue E47 had a similar effect on neurovirulence. Furthermore, the alkaline E138 residue enhanced susceptibility to heparin inhibition in vitro and limited JEV diffusion in mouse brain. These results suggest that the acidity/alkalinity of the E138 residue plays an important role in neurovirulence determination. IMPORTANCE The E protein is the only glycoprotein in mature JEV, and it plays an important role in viral neurovirulence. E protein mutations attenuate JEV neurovirulence through unclear mechanisms. Here, we discovered that E138 is a predominant determinant of JEV neurovirulence. We demonstrated that the alkalinity/acidity of E138 determines JEV neurovirulence. These data contribute to the characterization of the E protein and the rational development of novel JEV vaccines., (Copyright © 2018 American Society for Microbiology.)

Flaviviruses are major emerging human pathogens on a global scale. Some flaviviruses can infect the central nervous system of the host and therefore are regarded as neurotropic. The most clinically relevant classical neurotropic flaviviruses include Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. In this review, we focus on these flaviviruses and revisit the concepts of flaviviral neurotropism, neuropathogenicity, neuroinvasion, and resultant neuropathogenesis. We attempt to synthesize the current knowledge about interactions between the central nervous system and flaviviruses from the neuroanatomical and neuropathological perspectives and address some misconceptions and controversies. We hope that revisiting these neuropathological concepts will improve the understanding of flaviviral neuroinfections. This, in turn, may provide further guiding foundations for relevant studies of other emerging or geographically expanding flaviviruses with neuropathogenic potential, such as Zika virus and dengue virus, and pave the way for intelligent therapeutic strategies harnessing potentially beneficial, protective host responses to interfere with disease progression and outcome.

BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses., Competing Interests: The authors have declared that no competing interests.

Background: Overstimulation of glutamate receptors, especially neuronal N-methyl-D-aspartate receptor (NMDAR), mediates excitatory neurotoxicity in multiple neurodegenerative diseases. However, the role of NMDAR in the regulation of Japanese encephalitis virus (JEV)-mediated neuropathogenesis remains undisclosed. The primary objective of this study was to understand the function of NMDAR to JEV-induced neuronal cell damage and inflammation in the central nervous system., Methods: The effect of JEV-induced NMDAR activation on the progression of Japanese encephalitis was evaluated using the primary mouse neuron/glia cultures and a mouse model of JEV infection. A high-affinity NMDAR antagonist MK-801 was employed to block the activity of NMDAR both in vitro and in vivo. The subsequent impact of NMDAR blockade was assessed by examining the neuronal cell death, glutamate and inflammatory cytokine production, and JEV-induced mice mortality., Results: JEV infection enhanced the activity of NMDAR which eventually led to increased neuronal cell damage. The data obtained from our in vitro and in vivo assays demonstrated that NMDAR blockade significantly abrogated the neuronal cell death and inflammatory response triggered by JEV infection. Moreover, administration of NMDAR antagonist protected the mice from JEV-induced lethality., Conclusion: NMDAR plays an imperative role in regulating the JEV-induced neuronal cell damage and neuroinflammation. Thus, NMDAR targeting may constitute a captivating approach to rein in Japanese encephalitis.