Phospholipase A2 (PLA2) is a crucial enzyme in lipid metabolism and inflammatory signaling, playing a significant role in macrophage activation and neuroinflammation. However, its role in Japanese encephalitis virus (JEV)-mediated immune responses remains unclear. In this study, we show that PLA2 and its associated receptors, lysophosphatidic acid receptors (LPAR1 and LPAR3), are significantly upregulated at 24 h post-JEV infection, suggesting their involvement in JEV-induced inflammatory responses. To investigate the effect of PLA2 inhibition, we used arachidonyl trifluoromethyl ketone (AACOCF3). Molecular docking using AutoDock Vina 1.1.2, supported by Protein Data Bank and AlphaFold structures, indicated that AACOCF3 interacts with key inflammatory targets including gp91phox (− 7.9 kcal/mol) and LPAR3 (− 7.8 kcal/mol), suggesting its potential to modulate inflammatory and oxidative pathways. In vitro studies in JEV-infected macrophages demonstrated that AACOCF3 reduced activation of calcium-dependent cytosolic (c) PLA2 expression and phosphorylation, LPAR1, 3, MAPK signaling (JNK, ERK1/2, p38), and NF-κB protein levels, along with a reduction in JE viral RNA levels, likely reflecting indirect effects mediated through host pathway modulation. AACOCF3 also decreased proinflammatory cytokines (IL-1β, TNF-α) and autophagy-related markers (pAkt, LC3-II/I, SQSTM1/p62), and reduced expression of the ROS-generating enzyme gp91phox (NOX2), indicating partial attenuation of inflammatory and oxidative stress–associated pathways. Oxidative stress was assessed via gp91phox expression, and NF-κB was evaluated at protein level. These findings suggest that PLA2 contributes to JEV-induced inflammatory signaling, and that its inhibition modulates host responses during infection in vitro, rather than exerting a definitive therapeutic effect.

Skeletal muscle wasting is a clinically proven pathology associated with Japanese encephalitis virus (JEV) infection; however, underlying factors that govern skeletal muscle damage are yet to be explored. The current study aims to investigate the pathobiology of skeletal muscle damage using a mouse model of JEV infection. Our study reveals a significant increment in viral copy number in skeletal muscle post-JEV infection, which is associated with enhanced skeletal muscle cell death. Molecular and biochemical analysis confirms NOX2-dependent generation of reactive oxygen species, leading to autophagy flux inhibition and cell apoptosis. Along with this, an alteration in mitochondrial dynamics (change in fusion and fission process) and a decrease in the total number of mitochondria copies were found during JEV disease progression. The study represents the initial evidence of skeletal muscle damage caused by JEV and provides insights into potential avenues for therapeutic advancement.

The factors mitigating the microglia/macrophage activation and inflammatory damage in Japanese encephalitis (JE) virus infected CNS are still being ascertained. We aim to characterize the changes in iron transporter and iron storage proteins along with inflammatory and oxidative stress-mediated signaling during the JE viral infection. Cortical tissue samples from mice with JE viral infection were processed for biochemical, histological, and molecular analysis. Iron storage protein, i.e., ferritin, was found significantly increased post-JE viral infection, and iron accumulation was noted in cortical tissue. Key proinflammatory associated markers, such as TNF-α, IL-6, and its regulator TLR4, were found to be increased, while SOCS1 (anti-inflammatory regulator) transcription decreased with increased levels of oxidative stress markers NOX2-mediated NF-ΚB/p65 and protein carbonyl. Furthermore, it is noted that hepcidin level increased and ferroportin level decreased, and iron transporter gene expression got imbalanced after JE viral infection. This observation was further confirmed by deferoxamine (DFO) treatment to JE viral infection mice model, where the decline in hepcidin transcription level and iron load in cortical tissue of JE viral infected animals was noted. However, no change was found in the ferroportin level compared to JE viral infected animals. Together, these findings suggest that iron overload and hepcidin-ferroportin regulation are involved in JE viral infection disease pathologies and associated with the inflammatory and oxidative status of the host during infection.

Mitochondria dysfunction may be an important contributor to Japanese encephalitis (JE) viral infection disease pathogenesis. In the current study, we define whether changes in mitochondrial DNA copy number (which is the biomarker for mitochondrial function) and alteration in mitochondria dynamics (fusion and fission) contribute to the pathology of the JE virus in vivo mice model. We found decreased mitochondria copy number, reduced activation of mitochondrial fission (FIS1/DRP1), and increased activation of mitochondrial fusion (MFN1/MFN2/OPA1) genes that are associated with increased NOX2-mediated ROS generation and neuronal cell death following JE virus infection. Furthermore, we found that antioxidant glutathione level decreases. In summary, the following study demonstrates that JE viral infection causes an imbalance in mitochondrial fission/fusion gene activation and promotes NOX2-mediated oxidative stress and cell death, suggesting that intervention in mitochondrial dynamics might be a potential therapeutic strategy for combating oxidative stress and inflammatory process in JE viral infection.

Cerebellar dysfunction is implicated in impaired motor coordination and balance, thus disturbing the dynamics of sensorimotor integration. Neuroinflammation and aging could be prominent contributors to cerebellar aber-ration. Additionally, changes in mitochondrial dynamics may precede microglia activation in several chronic neurodegenerative diseases; however, the underlying mechanism remains largely unknown. Here using LPS (1 mg/kg i.p. for four consecutive days) stimulation in both young (3 months old) and aged (12 months old) mice, followed by molecular analysis on the 21st day, we have explored the correlation between aging and mito-chondrial dynamic alteration in the backdrop of chronic neuroinflammation. Following LPS stimulation, we observed microglia activation and subsequent elevation in proinflammatory cytokines (M1; TNF-alpha, IFN-gamma) with NLRP3 activation and a concomitant reduction in the expression of anti-inflammatory markers (M2; YM1, TGF-beta 1) in the cerebellar tissue of aged mice compared with the young LPS and aged controls. Remarkably, senes-cence (p21, p27, p53) and epigenetic (HDAC2) markers were found upregulated in the cerebellum tissue of the aged LPS group, suggesting their crucial role in LPS-induced cerebellar deficit. Further, we demonstrated alteration in the antagonistic forces of mitochondrial fusion and fission with increased expression of the mito-chondrial fission-related gene [FIS1] and decreased fusion-related genes [MFN1 and MFN2]. We noted increased mtDNA copy number, microglia activation, and inflammatory response of IL1-beta and IFN-gamma post-chronic neuro-inflammation in aged LPS group. Our results suggest that the crosstalk between mitochondrial dynamics and altered microglial activation paradigm in chronic neuroinflammatory conditions may be the key to under-standing the cerebellar molecular mechanism.

Integrated analysis of iron regulatory biomarkers and inflammatory response could be an important strategy for Japanese encephalitis viral (JEV) infection disease management. In the present study, the inflammatory response was assessed by measuring serum Interleukin-6 (IL-6) levels using ELISA, and the transcription levels of iron homeostasis regulators were analyzed via RT-PCR. Furthermore, inter-individual variation in the transferrin gene was analyzed by PCR–RFLP and their association with clinical symptoms, susceptibility, severity, and outcomes was assessed through binary logistic regression and classification and regression tree (CART) analysis. Our findings revealed elevated levels of IL-6 in serum as well as increased expression of hepcidin (HAMP), transferrin (TF), and transferrin receptor-1 (TFR1) mRNA in JEV infection cases. Moreover, we found a genetic variation in TF (rs4481157) associated with clinical symptoms of meningoencephalitis. CART analysis indicates that individuals with the wild-type TF genotype are more susceptible to moderate JEV infection, while those with the homozygous type are in the high-risk group to develop a severe JEV condition. In summary, the study highlights that JEV infection induces alteration in both IL-6 levels and iron regulatory processes, which play pivotal roles in the development of JEV disease pathologies.

Disorders of the central nervous system are worldwide causes of morbidity and mortality. Neurological disorders pose a large burden on worldwide health. The Global Burden of Disease Study shows that the neurological disorders such as Alzheimer's and other dementias, Parkinson's disease, multiple sclerosis, epilepsy, muscular dystrophy, and headache disorders represent 3% of the worldwide. Neurological disorders are defined as an inappropriate or impaired function of the peripheral or CNS due to impaired electrical impulses throughout the nervous system that may show the heterogeneous symptoms according to the parts of the system, which is involved in these pathologic processes. Some growing evidence on brain inflammation, viral or bacterial infection, and genetic components of neurological disease has been collected during recent years. On the basis of this evidence, we focused on the association of brain inflammation, viral or bacterial infection, mitochondrial gene, and other genetic components or mutations with neurological disorders.

Controlling the proinflammatory response of microglia by targeting chemokines (C-C motif) receptor 2 (CCR2) could be an important therapeutic approach for Japanese encephalitis virus (JEV) infection. Here, through JEV infection to BV2 microglia and young BALB/c mice, we investigated that CCR2 is highly upregulated after JEV infection and plays a key role in determining microglia activation phenotype and associated with neurotoxic proinflammatory mediators of TNF-α and IFNγ. In addition, we found JEV infection to BV2 microglia causes an increase in microglial proliferation and cell body area at day 1 and day 3. Using the agonist molecule of CCR2 inhibition; RS102895, significantly reduces microglia reactive phenotype and nitric oxide production. Further, to define the role of CCR2 in functional responses of microglia and their activation phenotype, we performed in vitro cell scratch functional assay and ImageJ analysis. When compared with control, microglia cells showed a significant increase in elongated or rod-like activated phenotype in JEV-infected cells at 24 h post-infection and CCR2 inhibition significantly reduced the elongated activation phenotype induced by JEV infection, suggesting that CCR2 acts as a critical regulator for microglia activation phenotype after JEV infection. We found that JEV-infected mice treated with RS102895 had less microglia activation and reduced mRNA expression of CCR2 and proinflammatory mediators such as IFN-γ in cortical tissue. Collectively, our data indicate that CCR2 drives reactive phenotype of microglia and its inhibition reduces microglia activation and neurotoxic proinflammatory mediators after JEV infection.

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.