Novel antiviral discoveries for Japanese encephalitis virus infections through reporter virus-based high-throughput screening.

Japanese encephalitis (JE) caused by JE virus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at-risk populations. This underscores the urgent need for prophylaxis and potent anti-JEV drugs. In this context, a high-content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high-throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA-dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti-JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.

Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein.

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

NS5-V372A and NS5-H386Y variations are responsible for differences in interferon α/ß induction and co-contribute to the replication advantage of Japanese encephalitis virus genotype I over genotype III in ducklings.

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.

Autophagy inhibitors alleviate Japanese encephalitis virus-induced cerebral inflammation in mice.

Japanese encephalitis (JE) is a zoonotic epidemic disease caused by Japanese encephalitis virus (JEV), and currently, no medicines are available to treat this disease. Autophagy modulators play an important role in the treatment of tumors, heart disease, and some viral diseases. The aim of this study was to investigate the effects of autophagy modulators on JEV infection and the host response in mice. The experimental mice were grouped as follows: DMEM (control), JEV, JEV+rapamycin (JEV+Rapa), JEV+wortmannin (JEV+Wort), JEV+chloroquine (JEV+CQ), Rapa, Wort, and CQ. The control group was treated with DMEM. The mice in other groups were infected with 105 PFU of JEV, and Rapa, Wort, and CQ were administered 2 h prior to JEV challenge and then administered daily for 10 consecutive days. All mice were monitored for neurological signs and survival. The damage of subcellular structures in the mouse brain was evaluated by transmission electron microscopy. The distribution of virus in the mouse brain was determined by RNAScope staining and immunohistochemical staining. The neuroinflammatory responses in the brain were examined via quantitative real-time PCR, and the signal pathways involved in neuroinflammation were identified by Western blot. The mice in the JEV+Wort and JEV+CQ groups showed milder neurological symptoms, less damage to the mitochondria in the brain tissue, and a higher survival rate than those in the JEV+Rapa and JEV groups. Compared with the JEV+Rapa and JEV groups, the distribution of JEV in the brain of mice in the JEV+Wort and JEV+CQ groups was lower, and the inflammatory response was weaker. No significant difference was observed in the expression of the PI3K/AKT/NF-κB pathway in mouse brain among the different groups. Our study suggests that the autophagy inhibitors Wort and CQ reduce JEV infection and weaken the inflammatory response, which does not depend on the PI3K/AKT/NF-κB pathway in mouse brain.

Efficacy and safety of intravenous high-dose immunoglobulin in treatment of the severe form of Japanese encephalitis.

PURPOSE: To explore the efficacy and safety of intravenous immunoglobulin (IVIG) in the treatment of severe Japanese encephalitis (JE). METHODS: A retrospective study of 124 children diagnosed with the severe or very severe form of JE was undertaken. There were 62 cases in the IVIG group and control group. The efficacy, safety, and tolerability of IVIG were evaluated 3 days, as well as 1, 2, and 3 weeks after IVIG, respectively, and the prognosis was assessed at 6 months. RESULTS: Cox regression survival analysis suggested that the IVIG group reached the criteria for efficacious treatment faster than that in the control group. The duration of unconsciousness and the number of days of seizures, a dull response to light, the pyramidal sign, and meningeal-irritation sign in the IVIG group were shorter than those in the control group (p < 0.05). The number of complications occurring in the IVIG group (including gastrointestinal bleeding and pneumonia) was less than that in the control group (p < 0.05). Cox regression survival analysis suggested that age (p = 0.003) and imaging abnormalities (p = 0.042) had an effect on the efficacy of IVIG treatment. The Liverpool Outcome Score at 6 months showed that the prognosis of the IVIG group was better than that of the control group (p < 0.05). IVIG treatment was safe and tolerable. CONCLUSION: IVIG showed good efficacy, safety, and tolerance for treatment of the severe form of JE. The age and imaging abnormalities of patients affect the efficacy of IVIG treatment.

RNA Interference Screening Reveals Requirement for Platelet-Derived Growth Factor Receptor Beta in Japanese Encephalitis Virus Infection.

Mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide and is associated with high morbidity and mortality. To identify potential host therapeutic targets, a high-throughput receptor tyrosine kinase small interfering RNA library screening was performed with recombinant JEV particles. Platelet-derived growth factor receptor beta (PDGFRß) was identified as a hit after two rounds of screening. Knockdown of PDGFRß blocked JEV infection and transcomplementation of PDGFRß could partly restore its infectivity. The PDGFRß inhibitor imatinib, which has been approved for the treatment of malignant metastatic cancer, protected mice against JEV-induced lethality by decreasing the viral load in the brain while abrogating the histopathological changes associated with JEV infection. These findings demonstrated that PDGFRß is important in viral infection and provided evidence for the potential to develop imatinib as a therapeutic intervention against JEV infection.

miR-301a Regulates Inflammatory Response to Japanese Encephalitis Virus Infection via Suppression of NKRF Activity.

Microglia being the resident macrophage of brain provides neuroprotection following diverse microbial infections. Japanese encephalitis virus (JEV) invades the CNS, resulting in neuroinflammation, which turns the neuroprotective role of microglia detrimental as characterized by increased microglial activation and neuronal death. Several host factors, including microRNAs, play vital roles in regulating virus-induced inflammation. In the current study, we demonstrate that the expression of miR-301a is increased in JEV-infected microglial cells and human brain. Overexpression of miR-301a augments the JEV-induced inflammatory response, whereas inhibition of miR-301a completely reverses the effects. Mechanistically, NF-κB-repressing factor (NKRF) functioning as inhibitor of NF-κB activation is identified as a potential target of miR-301a in JEV infection. Consequently, miR-301a-mediated inhibition of NKRF enhances nuclear translocation of NF-κB, which, in turn, resulted in amplified inflammatory response. Conversely, NKRF overexpression in miR-301a-inhibited condition restores nuclear accumulation of NF-κB to a basal level. We also observed that JEV infection induces classical activation (M1) of microglia that drives the production of proinflammatory cytokines while suppressing alternative activation (M2) that could serve to dampen the inflammatory response. Furthermore, in vivo neutralization of miR-301a in mouse brain restores NKRF expression, thereby reducing inflammatory response, microglial activation, and neuronal apoptosis. Thus, our study suggests that the JEV-induced expression of miR-301a positively regulates inflammatory response by suppressing NKRF production, which might be targeted to manage viral-induced neuroinflammation.

Screening of Natural Extracts for Inhibitors against Japanese Encephalitis Virus Infection.

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.

Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus.

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

Japanese Encephalitis Virus-Induced Anti-N-Methyl-D-Aspartate Receptor Encephalitis: A Case Report and Review of Literature.

Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARe) was originally described as a paraneoplastic disease with more than 50% cases involving a tumor. However, tumor incidence in anti-NMDARe in children is much lower. Herpes simplex virus-induced anti-NMDARe has been well-described; however, findings on Japanese encephalitis virus (JEV)-induced anti-NMDARe are scarce. Here, we describe a 7-year-old boy who presented with fever and headache that progressed to seizures and disturbance of consciousness. Brain magnetic resonance imaging (MRI) revealed abnormalities in the bilateral globus pallidus. The diagnosis of JE was made based on a positive JE antibody test results in serum and cerebrospinal fluid. Antiviral and symptomatic therapies led to rapid recovery. Four weeks after the onset of JE, the patient presented with emotional and behavioral disturbances, sleep difficulty, and extrapyramidal symptoms. MRI showed symmetrical lesions in the bilateral thalami and basal ganglia which were expanded than those on the original scan. Antibodies against NMDAR were detected and immunotherapy led to significant recovery. This case and our literature review suggest that JEV may be a clinically important cause of anti-NMDARe in children. Patients with JE-induced anti-NMDARe present with symptoms similar to those of patients with primary anti-NMDARe. Most patients with JE-induced anti-NMDARe showed a good response to first-line immunotherapies.

Activation of neuronal N-methyl-D-aspartate receptor plays a pivotal role in Japanese encephalitis virus-induced neuronal cell damage.

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.

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca2+ channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.

Chemokine receptor antagonist block inflammation and therapy Japanese encephalitis virus infection in mouse model.

Japanese encephalitis (JE) is a viral encephalitis disease caused by infection with the Japanese encephalitis virus (JEV). The virus can cross the blood-brain barrier and cause death or long-term sequela in infected humans or animals. In this study, we first investigated the distribution of JEV infection in brain and further analyzed the dynamic change in inflammation related genes, chemokines, as well as pathological characteristics. Results demonstrated that CCR2 and CCR5 antagonist could significantly inhibit the inflammation. The mice treated with CCR2 and CCR5 antagonists had a higher survival rate between 60% and 70%, respectively. In summary, our study thoroughly illustrated the characteristics of the dynamic change in inflammation related genes and chemokines induced by JEV infection. We further indicated that CCR5 and CCR2 are potential targets for treatment of JE.

Drug Repurposing for Japanese Encephalitis Virus Infection by Systems Biology Methods.

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.

MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

UNLABELLED: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis. Albeit microRNAs (miRNAs) have emerged as major regulatory noncoding RNAs with profound effects on inflammatory response, it is unknown how astrocytic miRNAs regulate JEV-induced inflammation. Here, we found the involvement of miR-19b-3p in regulating the JEV-induced inflammatory responsein vitroandin vivo The data demonstrated that miR-19b-3p is upregulated in cultured cells and mouse brain tissues during JEV infection. Overexpression of miR-19b-3p led to increased production of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6, interleukin-1ß, and chemokine (C-C motif) ligand 5, after JEV infection, whereas knockdown of miR-19b-3p had completely opposite effects. Mechanistically, miR-19b-3p modulated the JEV-induced inflammatory response via targeting ring finger protein 11, a negative regulator of nuclear factor kappa B signaling. We also found that inhibition of ring finger protein 11 by miR-19b-3p resulted in accumulation of nuclear factor kappa B in the nucleus, which in turn led to higher production of inflammatory cytokines.In vivosilencing of miR-19b-3p by a specific antagomir reinvigorates the expression level of RNF11, which in turn reduces the production of inflammatory cytokines, abrogates gliosis and neuronal cell death, and eventually improves the survival rate in the mouse model. Collectively, our results demonstrate that miR-19b-3p positively regulates the JEV-induced inflammatory response. Thus, miR-19b-3p targeting may constitute a thought-provoking approach to rein in JEV-induced inflammation. IMPORTANCE: Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in humans worldwide. The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. Accumulating data indicate that miRNAs regulate a variety of cellular processes, including the host inflammatory response under pathological conditions. Recently, a few studies demonstrated the role of miRNAs in a JEV-induced inflammatory response in microglia; however, their role in an astrocyte-derived inflammatory response is largely unknown. The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells. Moreover, administration of an miR-19b-3p-specific antagomir in JEV-infected mice reduces neuroinflammation and lethality. These findings suggest a new insight into the molecular mechanism of the JEV-induced inflammatory response and provide a possible therapeutic entry point for treating viral 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.

Role of oral Minocycline in acute encephalitis syndrome in India - a randomized controlled trial.

BACKGROUND: Acute encephalitis syndrome (AES) is a public health problem in India. Neuroinfections are believed to be the most important etiology. Minocycline is a semisythetic tetracycline having excellent penetration into cerebrospinal fluid, established neuroprotective and antiviral properties besides action on nonviral causes of AES. It has been shown to be effective in animal model of Japanese encephalitis (JE). A randomized, controlled trial of nasogastric/oral minocycline in JE and AES at a single centre in Uttar Pradesh, northern India, was therefore conducted. METHODS: Patients beyond 3 years of age - but excluding women aged 16-44 years - hospitalized with AES of < =7 days duration were enrolled and block randomized to receive nasogastric/oral minocycline or placebo suspension and followed up. Patients, study personnel and those entering data were blinded as to drug or placebo received. Primary outcome was cumulative mortality at 3 months from hospitalization. Analysis was by intention to treat. RESULTS: 281 patients were enrolled, 140 received drug and 141 placebo. While there was no overall statistically significant difference in 3 month mortality between drug and placebo groups [RR = 0 · 83 (0 · 6-1 · 1)], there were encouraging trends in patients older than 12 years [RR = 0.70 (0.41-1.18)] and in Glasgow Outcome Score (GOS) at 3 months (χ(2) = 7 · 44, p = 0 · 059). These trends were further accentuated if patients dying within one day of reaching hospital were excluded [OR for 3 month mortality =0 · 70 (0 · 46-1 · 07), p = 0.090; 3 month GOS p = 0 · 028]. CONCLUSIONS: A trend towards better outcomes was observed with minocycline, especially in those patients who survived the initial day in hospital. These findings should form the basis for planning a larger study and possibly including minocycline in the initial management of AES as seen here. TRIAL REGISTRATION: The trial was registered with Clinical Trials Registry of India (CTRI) - CTRI/2010/091/006143.

Immunogenicity & safety of a single dose of live-attenuated Japanese encephalitis vaccine SA 14-14-2 in adults.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) caused by mosquito-borne Flavivirus is one of the leading causes of viral encephalitis in Asia. Control strategies include vector control and human vaccination. Due to lack of immunization programmes in endemic regions, there are still high mortality and morbidity. A live-attenuated SA 14-14-2 JE vaccine (LAJEV) has been licensed and used in Asian countries, including India. We report the assessment of immunogenicity and safety of the vaccine in adults during the first mass adult vaccination campaign carried out in Assam, India. METHODS: One thousand and seventy five adults (aged ≥15 yr) who received LAJEV were monitored for adverse events following immunization for one year. The safety assessment of vaccinated population was evaluated till 28 days and at 6 and 12 months. Blood samples collected from the enrolled participants were tested by plaque reduction neutralization test (PRNT 50 ) to assess the neutralizing antibody titres (NATs) before vaccination and 28 days, six and 12 months post-vaccination (PV). RESULTS: Among the 1075 vaccinated individuals, four reported minor adverse effects from 30 min to 28 days PV. Based on the pre-vaccination NAT, the study participants were categorized as seronegative, moderately seropositive and strongly seropositive. Nearly 85.5 per cent of JE seronegative participants seroconverted by 28 days PV. The geometric mean titre (GMT) in all the three groups increased by 28 days and decreased by six and 12 months PV. Nearly 60 per cent of the moderately positive individuals exhibited four-fold rise in GMT, 28 days PV. Almost 95.5 per cent of the participants in the study population remained seroprotected at the end of 12 months PV. INTERPRETATION & CONCLUSIONS: This study on immunogenicity and safety of LAJEV in adults showed that a single dose of the live-attenuated vaccine was safe and induced protective immunity to both JE seronegative and naturally seropositive adults. Further study is required to find out long term protective efficacy of this vaccine.