Japanese Encephalitis Virus infection induces inflammation of swine testis through RIG-I-NF-ĸB signaling pathway.

Japanese Encephalitis Virus (JEV) is an important zoonotic flavivirus transmitted by mosquitos. JEV infection in sows primarily manifests as a reproductive disease such as abortion and transient infertility while in infected boars, it can cause orchitis. Previous studies mainly focused on the pathogenesis of human encephalitis caused by JEV infection, while few concentrations have been made to unveil the potential mechanism of reproductive dysfunction in JEV-infected pigs. In this study, histopathological analysis and immunohistochemistry staining was performed on testis of JEV-infected boars, indicating that JEV could infect testicular cells and cause inflammatory changes in testis. In vitro assays reveal that primary swine testicular cells and swine testis (ST) cells are highly permissive to JEV and significant inflammatory response was shown during JEV infection. Mechanically, we found that JEV infection increases the expression of retinoic acid-inducible gene I (RIG-I) and activates transcription factor NF-κB. Production of pro-inflammatory cytokines was greatly reduced in JEV infected testicular cells after knockout of RIG-I or treatment with the NF-κB specific inhibitor. In addition, activation of NF-κB was also significantly suppressed upon RIG-I knockout. Taken together, our results reveal that JEV could infect boar testicles, and RIG-I-NF-κB signaling pathway is involved in JEV-induced inflammation in swine testicular cells.

Chronic post-encephalitic epilepsy following Japanese encephalitis: Clinical features, neuroimaging data, and outcomes.

PURPOSE: Japanese encephalitis (JE), the main cause of viral encephalitis in Asia, usually presents with acute symptomatic seizures; however, there have been very few systematic reports regarding late unprovoked seizures and epilepsy. We aimed to describe the clinical features and outcomes of post-encephalitic epilepsy following JE. METHODS: Patients with epilepsy with a previous confirmed diagnosis of JE visiting West China Hospital from 2013 to 2019 were enrolled in the observational case-controlled study. Patients with epilepsy with a history of other non-specific viral encephalitis were enrolled as controls. For all enrolled subjects, disease related information was recorded. RESULTS: Forty-eight patients with JE (20 males; median age, 21.0 years; average epilepsy duration, 8.55 years) were identified. The median duration from JE to the first unprovoked seizure was 7.73 years, which significantly differed from that of the controls (7.73 vs. 2.69 years, respectively; p = 4.59 × 10-6). Most patients had focal epilepsy, and 29 (78.38%) were drug resistant. Among 45 patients with available neuroimaging data, three in fourth had no obvious abnormality, and the temporal lobe and hippocampus (22.22%) were the most affected brain regions. Six patients had surgery, and three achieved class-one seizure-free status. CONCLUSION: The latency to the first unprovoked seizure was longer in patients with JE than controls. Regarding chronic epilepsy, three in four had structural abnormalities, and the long-term outcomes of post-encephalitic epilepsy following JE were poor. Surgery remains an option for drug-resistant epilepsy.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

A study of glutamate levels, NR1, NR2A, NR2B receptors and oxidative stress in rat model of Japanese encephalitis.

There is paucity of studies on the role of glutamate excitotoxicity in cell damage in Japanese encephalitis. In this study the glutamate levels and its NMDA receptors, and oxidative stress markers in different brain regions have been evaluated and correlated with neurobehavioral changes at different time points. Twelve day old Wistar rats were inoculated with 3×106pfu/ml intracerebrally. The neurobehavioral effects were evaluated by spontaneous locomotor activity (SLA), grip strength and rota rod test on 10, 33 and 48days post inoculation (dpi). Glutamate level was evaluated by enzyme linked immunosorbent assay, mRNA gene expression of ionotropic glutamate receptors N-methyl d-aspartate (NMDA) receptor 1, 2A and 2B (NR1, NR2A and NR2B) were evaluated by real time PCR. Malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx) levels were measured by spectrophotometer in different brain regions of JEV infected rats on 10, 33 and 48dpi. There was significant increase in motor deficit, grip strength and decreased locomotor activity on 10 and 33dpi. Glutamate levels were increased in thalamus, midbrain, frontal cortex, striatum and cerebellum on 10 and 33dpi and were followed by a recovery on 48dpi. Glutamate NMDR receptors NR1, NR2A and NR2B were reduced in thalamus, midbrain, frontal cortex, striatum and cerebellum on 10dpi which was followed by recovery after 33dpi. A significant increase in MDA level in thalamus, midbrain, frontal cortex, striatum and cerebellum was noted on 10 and 33dpi. The antioxidant GSH and GPx were significantly reduced in these brain regions on 10 and 33dpi. Glutamate, MDA, GSH and GPx correlated in different brain regions as the disease progress. Increased Glutamate level may be related to oxidative stress and may be responsible for behavioral alterations in rat model of Japanese encephalitis.

Bacillus Calmette-Guérin Confers Neuroprotection in a Murine Model of Japanese Encephalitis.

OBJECTIVE: Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. METHODS: Suckling mice were inoculated with 106 CFU of BCG and at 18 days postinoculation were challenged with 100 LD50 of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. RESULTS: The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. CONCLUSION: These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.

Overview: Japanese encephalitis.

Japanese encephalitis (JE) is one of the most important endemic encephalitis in the world especially in Eastern and Southeastern Asia. JE affects over 50,000 patients and results in 15,000 deaths annually. JE virus is a single stranded positive sense RNA virus belonging to family flaviviridae. JE virus is transmitted through a zoonotic cycle between mosquitoes, pigs and water birds. Humans are accidentally infected and are a dead end host because of low level and transient viremia. In the northern region, large epidemics occur during summers whereas in the southern region JE tends to be endemic: cases occur throughout the year with a peak in the rainy season. Occurrence of JE is more closely related to temperature than to humidity. JE is regarded as a disease of children in the endemic areas but in the newly invaded areas, it affects both the adults and children because of the absence of protective antibodies. For every patient of JE, there are large numbers of subclinical cases (25-1000). Symptomatic JEV infection manifests with nonspecific febrile illness, aseptic meningitis or encephalitis. Encephalitis manifests with altered sensorium, seizures and focal neurological deficit. Acute flaccid paralysis may occur due to anterior horn cell involvement. A wide variety of movement disorders especially transient Parkinsonian features and dystonia (limb, axial, orofacial) are reported in 20-60% patients. JE mainly affects thalamus, corpus striatum, brainstem and spinal cord as revealed by MRI and on autopsy studies. Coinfection of JE and cysticercosis occurs because of the important role of pigs in the life cycle of both JEV and cysticercosis. Laboratory diagnosis of JE is by IgM capture ELISA, which has high sensitivity and specificity. In the absence of specific antiviral therapy, JE is managed by symptomatic and supportive therapies and preventive measures. Purified formalin inactivated mouse brain derived vaccine and live attenuated vaccine (SA 14-14-2) are available; the latter is reported to be safe, effective and cheap. The role of Chimeric recombinant attenuated JE vaccine is under investigation. Control of JE is related to the wider issues of hygiene, environment, education and economy.

Understanding the molecular mechanism of blood-brain barrier damage in an experimental model of Japanese encephalitis: correlation with minocycline administration as a therapeutic agent.

The blood-brain barrier (BBB) serves to protect the central nervous system (CNS) from damage by exogenous molecules. Japanese encephalitis (JE), caused by a neurotropic flavivirus, leads to inflammation in the CNS, neuronal death and also compromises the structural and functional integrity of the BBB. Minocycline, a semisynthetic tetracycline, has been found to be broadly protective in neurological disease models featuring inflammation and cell death and at present, is being evaluated in clinical trials. In the present study, we propose that the neuroprotective role of minocycline in experimental models of JE extends also to the protection of the BBB. Damage to the BBB was assessed by Evan's blue dye exclusion test after minocycline treatment following Japanese encephalitis virus (JEV) infection. A breakdown of the BBB occurred in mice inoculated intravenously with JEV. This resulted in leakage of protein-bound Evan's blue dye into the brain tissue. Semi-quantitative RT-PCR revealed an up-regulation of chemokine receptors and adhesion molecules following JEV infection. Immunostaining showed leukocyte and neutrophil infiltration following JEV infection. Intraperitoneal injection of minocycline, beginning 24h post-JEV infection, abrogated the effects by reducing BBB damage, decreasing expression of iNOS, Cox-2, VEGF and also by reducing the elevated level of transcript of chemokine receptors and adhesion molecules in the brain. Matrix metalloproteinases (MMPs) are known to disrupt the BBB and minocycline was found to significantly decrease the activity of MMP-9 in brain tissue homogenates. Thus, minocycline, administered at a clinically relevant time, appears to maintain blood-brain barrier integrity following JEV infection.

Japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines.

Astrocytes become activated in response to many CNS pathologies. The process of astrocyte activation remains rather enigmatic and results in so-called reactive gliosis, a reaction with specific structural and functional characteristics. Astrocytes play a vital role in regulating aspects of inflammation and in the homeostatic maintenance of the CNS. However, the responses of different human astroglial cell-lines in viral encephalitis mediated inflammation are not well documented. We have shown that Japanese encephalitis virus (JEV) infection causes morphological and functional changes in astrocytic cell-lines. We have demonstrated that besides reactive oxygen species (ROS) JEV infection differentially regulated the induction pattern of IL-6, IL-1 beta and IL-8. IP-10, MCP-1, MIG and RANTES secretions in different astroglial cell-lines. The expression of different proteins such as astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter/essential amino acid transporter-1 (GLAST/EAAT-1), glutamate transporter-1/essential amino acid transporter-2 (GLT-1/EAAT-2), Ceruloplasmin and Thioredoxin (TRX) expression level also differ in different human astrocyte cell-lines following infection.

Japanese encephalitis virus infection stimulates Src tyrosine kinase in neuron/glia.

Japanese encephalitis virus (JEV) is a neurotropic virus. The clinically manifestation of JEV-induced encephalitis is characterized by the brain inflammation and neuronal dysfunction and/or destruction. Currently, the cellular signaling molecules that underlie JEV-induced cerebral inflammation and cellular alterations are not well understood. Protein tyrosine phosphorylation events are key regulators of cellular signaling processes, including inflammation. We investigated whether Src protein tyrosine kinase (PTK) function in JEV-induced cellular changes in neuron/glia cultures. JEV infection modulated tyrosine phosphorylation events. Src PTK was hyperphosphorylated at the early stage of infection. Biochemical studies demonstrated that both inhibitors of the Src family PTK and Ras attenuated JEV-induced extracellular signal-regulated kinase (ERK) activation. Our results further revealed that PTK, Ras, and ERK inhibitors effectively suppressed JEV-induced pro-inflammatory cytokine expression and neurotoxicity. Pharmacological studies suggested that microglia secreted pro-inflammatory cytokine via Src/Ras/ERK pathway in responding to JEV infection. Another interesting observation was that nonstructural protein 3 (NS3) was able to interact with Src and showed tyrosine phosphorylation. However, the biological consequences of their interaction and exact control of NS3 tyrosine phosphorylation required further investigation. Our results suggest that the Src/Ras/ERK signaling cascade is involved in JEV-induced pro-inflammatory cytokine expression and neurotoxicity.

Japanese encephalitis virus infection decrease endogenous IL-10 production: correlation with microglial activation and neuronal death.

The anti-inflammatory cytokine interleukin (IL)-10 is synthesized in the central nervous system (CNS) and acts to limit clinical symptoms of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioral changes that occur during bacterial infections. Expression of IL-10 is critical during the course of most major diseases in the CNS and promotes survival of neurons and all glial cells in the brain by blocking the effects of proinflammatory cytokines and by promoting expression of cell survival signals. In order to assess functional importance of this cytokine in viral encephalitis we have exploited an experimental model of Japanese encephalitis (JE). We report for the first time that in Japanese encephalitis, there is a progressive decline in level of IL-10. The extent of progressive decrease in IL-10 level following viral infection is inversely proportional to the increase in the level of proinflammatory cytokines as well as negative consequences that follows viral infection.

A comparison of clinical and radiological findings in adults and children with Japanese encephalitis.

BACKGROUND: Japanese encephalitis (JE) is the most common human endemic encephalitis, prevalent mainly in Southeast Asia. It affects both adults and children in different areas, but there is no comparative study of their clinical features and outcomes. OBJECTIVE: To evaluate clinical and radiological features in adults and children with JE. METHODS: Patients with serologically or virologically confirmed JE who were treated during the past 10 years were included in this study. All patients underwent a detailed neurological examination, computed tomography, or magnetic resonance imaging. The presence of movement disorders, anterior horn cell involvement, and electroencephalographic changes was noted. After 6 months, each patient's outcome was defined as poor, partial, or complete recovery. The clinical and radiological findings for both adults and children were compared using chi2 tests. RESULTS: The results are based on 30 children and 37 adults. Seizure was present in 23 adults (62.2%) and in 17 children (56.7%). Three children had associated neurocysticercosis, and all of them had partial seizures. The occurrence of focal neurological deficit, anterior horn cell involvement, and parkinsonian features was not significantly different between adults and children. Dystonia was more common in children, occurring in 20 (66.7%) compared with 7 adults (18.9%). Six adults died, but none of the children did; however, the 6-month outcome was better for surviving adults compared with the children. Computed tomography and magnetic resonance imaging findings were not significantly different between the 2 groups. CONCLUSIONS: Children with JE are more likely to have dystonia and a poor outcome at 6 months compared with adults. The difference in clinical findings and outcome in children and adults with JE may be owing to immunological factors, maturation of the central nervous system, and neuronal plasticity.

Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines.

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met-->Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.

Correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in Japanese encephalitis patients.

To investigate the prognostic role of tumour necrosis factor (TNF) in Japanese encephalitis virus (JEV) infection, we measured the immunoreactive forms of TNF concentrations in the serum and cerebrospinal fluid (CSF) of 47 laboratory-confirmed cases of JE. It was observed that TNF levels were elevated (> 15 pgm/ml) in all the 47 serum samples (range 19.4-923.8 pg/ml), while in 46/47 CSF samples TNF was elevated (range 10.8-376 pg/ml). The mean (SD) TNF levels in the serum of fatal cases was 234.34 pg/ml (304.40) as compared to the mean of 85.31 pg/ml (SD 153.92) in nonfatal cases. Similar observations were also made with respect to the TNF levels in the CSF; the mean of fatal cases was 69.39 pg/ ml (SD 39.00) in contrast to the mean of 62.41 pg/ml (SD 75.25) of nonfatal cases. The increase in TNF levels did not show any correlation to the duration of illness. It was further observed that the mortality rate increased with increasing concentrations of TNF in the serum and CSF. Correlation of laboratory parameters to final outcome revealed that TNF concentrations above 50 pg/ ml in serum correlated significantly (P = .05) with a fatal outcome, whilst high levels of JEV-IgM antibodies (> 500 units) in the CSF correlated with a nonfatal outcome (P = .03). These results suggest that TNF can be used as a possible prognosticator of a fatal outcome in JEV infection.

Regulation of vascular permeability by macrophage-derived chemotactic factor produced in Japanese encephalitis.

The vascular effects of Japanese encephalitis virus (JEV)-stimulated splenic macrophage-derived neutrophil chemotactic factor (MDF) were evaluated in mice. Intraperitoneal injection of MDF in mice resulted in a rapid increase in capillary permeability in a dose-dependent manner as assessed by leakage of intravenously injected radiolabelled albumin ([125I]-albumin) or Evans blue dye. Intradermal inoculation of MDF in rabbits caused [51Cr]-labelled neutrophil emigration and accumulation into injected sites. Peak plasma leakage and neutrophil infiltration were observed at 1 h following MDF inoculation, and plasma leakage was restored by 2.5 h. The increase in capillary permeability was sensitive to pretreatment of mice with avil and ranitidine (H1 and H2 histamine receptor blockers, respectively), resulting in abrogation of the response; indomethacin, a prostaglandin synthetase inhibitor, did not have any effect.

Effect of cadmium on Japanese encephalitis virus infection in mice. 1. Acute and single-dose exposure experiment.

When mice were treated with 0.09 mg cadmium chloride (Cd) per mouse once and inoculated i.p. simultaneously with Japanese encephalitis virus (JEV) they showed a significant difference in the incubation time and mortality between the treated and untreated groups in repeated experiments. Cd treatment shortened the incubation time and the mortality increased greater than twice compared with the untreated control. This effect was not observed in the case of intracerebral inoculation of JEV. Effects of Cd on antibody formation in mice were also determined. Animals given a single s.c. dose of Cd were immunized with JE inactivated vaccine once simultaneously. When mice were treated with Cd, they did not show low neutralizing and hemagglutination inhibition activities compared with the control mice. Pretreatment of Cd did not affect any mortality or antibody formation.