Japanese Encephalitis Virus Infection Results in Transient Dysfunction of Memory Learning and Cholinesterase Inhibition.

Cholinergic system has an important role in memory and learning. Abnormal cognitive and behavioral changes have been reported in Japanese encephalitis (JE), but their basis has not been comprehensively evaluated. In this study, we report memory and learning and its association with acetylcholinesterase (AChE) activity, JE virus titer, and with histopathological observations in a rat model of JE. Wistar rats were intracerebrally inoculated on 12th day with 3 × 106 pfu/ml of JE virus. Memory and learning were assessed by the active and passive avoidance tests on 10, 33, and 48 days post inoculation (dpi). After 10, 33, and 48 dpi AChE activity, Japanese encephalitis virus (JEV) titer and histopathological changes were studied in the frontal cortex, thalamus, midbrain, cerebellum, and hippocampus. There was significant impairment in memory and learning on 10 dpi which started improving from 33 dpi to 48 dpi by active avoidance test. Passive avoidance test showed decrease in transfer latency time of retention trial compared to acquisition on first, second, and third retention day trial compared to controls. AChE inhibition was more marked in the hippocampus, frontal cortex, and cerebellum on 10 dpi. However, AChE activity started improving from 33 dpi to 48 dpi. AChE activity in the thalamus and midbrain correlated with active avoidance test on 10 dpi and 33 dpi. Histopathological studies also revealed improvement on 33 and 48 compared to 10 dpi. The present study demonstrates transient memory and learning impairment which was associated with reduction in AChE, JEV titer, and damage in different brain regions of JEV infected rats.

Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

BACKGROUND: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. METHODS: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed. RESULTS: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment. CONCLUSIONS: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions.

Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5' and 3' un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections.

Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication.

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

Upregulated expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in BALB/c mouse brain challenged with Japanese encephalitis virus.

BACKGROUND: Uncontrolled immune responses in the nervous system are potentially damaging following Japanese encephalitis virus (JEV) infection. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) act together to control the proteolysis of extracellular matrix. Disbalances in the MMP/TIMP system during virally induced neurodegenerative processes and inflammations are responsive to changes in the progression of diseases. METHODS: The expression of MMP-2, MMP-7, MMP-9, TIMP-1, and TIMP-3 in JEV-infected mouse brain was analyzed by RT-PCR for semiquantitation and ELISA for estimation of protein along with brain histopathology at different days postinoculation (dpi). Gelatin gel zymography was performed for MMP-2 and MMP-9 activities. RESULTS: In the virus-infected group, expression of MMP-2, MMP-7, MMP-9, TIMP-1, and TIMP-3 was found to be increased from 1 dpi to 6 dpi as compared to controls by both RT-PCR and ELISA. The expressions of MMPs and TIMPs at mRNA and protein levels were in concordance with each other. Post hoc multiple comparison analysis between days revealed that, in the virus-infected groups, significant increases (p < 0.05) in MMP and TIMP levels were observed between various dpi at both mRNA and protein levels. Only the MMP-7 protein level at 6 dpi was not significant compared to 5 dpi (p = 0.99). CONCLUSION: Overexpression of MMPs and TIMPs is associated with disease severity in the central nervous system (CNS) during JEV infection. Our results showed that JEV infection can alter the expression of MMPs and TIMPs in the CNS. Thus, assessing these important immune mediators in CNS infection appears to play an important role in the development of symptoms and may help to understand the JEV-induced neurological disorders. More studies are required on this important enzymatic system to study their role in immune mediated pathogenesis.

Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha.

We have overexpressed an 8.5-kDa mouse Ca(2+)/calmodulin kinase II inhibitor alpha protein (mCaMKIINalpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca(2+)/calmodulin kinase II (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIINalpha react with an approximately 37-kDa protein present in mouse brain. The pattern of expression of the approximately 37-kDa protein is similar to that of mCaMKIINalpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the approximately 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKIIalpha is known to perform key regulatory functions. We conclude that the approximately 37-kDa protein identified in this study is mCaMKIINalpha and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKIIalpha.

Regulation of Ca2+/calmodulin kinase II inhibitor alpha (CaMKIINalpha) in virus-infected mouse brain.

The alpha and beta isoforms of rat Ca(2+)/calmodulin kinase II inhibitor (CaMKIINalpha/beta) expressed in brain or brain and testis, respectively, are potent inhibitors of Ca(2+)/calmodulin kinase II (CaMKII) in vitro. However, the regulation or function of CaMKIINalpha/beta in the central nervous system (CNS) is not known. In this study, we demonstrate that mouse CaMKIINalpha gene encodes a 2.9kb brain-specific transcript whose expression is downregulated in mouse brain during Japanese encephalitis virus (JEV) and rabies virus infection. The downregulation is specific for CaMKIINalpha but not CaMKIINbeta mRNA. In addition to these changes in CaMKIINalpha mRNA, distinct changes are also observed in the phosphorylation as well as subcellular localization of CaMKIIalpha leading to an increase in cytosolic CaMKII activity in JEV-infected mouse brain. The differential regulation of CaMKIIalpha and CaMKIINalpha during JEV infection suggests a possible role for these proteins in viral infection and/or virus-induced neuropathogenesis in the CNS.

Polymorphisms of the gene coding for copper/zinc superoxide dismutase (SOD1) in patients with Japanese encephalitis.

Japanese encephalitis is the commonest form of encephalitis globally. Most cases develop characteristic encephalitis but some also present with flaccid paralysis. The paralysis is secondary to damage at the alpha motor neurone, the site that is also damaged in amyotrophic lateral sclerosis (ALS). The gene coding for superoxide dismutase 1 (SOD1) is thought to be involved in ALS and may also be linked to susceptibility to Japanese encephalitis. To investigate this possibility, polymorphisms in the SOD1 gene were investigated, in 61 cases of Japanese encephalitis, 61 matched controls and 171 population controls, in Vietnam. Novel polymorphisms, found only in three of the cases and one of the population controls, may be involved with susceptibility to Japanese encephalitis and potentially to other flavivirus infections that lead to damage to the cells of the anterior horn. Further research on this possible association is required.

Japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures.

Japanese encephalitis virus (JEV) infection is commonly associated with inflammatory reaction and neurological disease that occurs in the infected animals. Reactive oxygen species have been implicated as a critical mediator for inflammation and diseases. The present study investigated the change of redox potential in glial cells following JEV infection. JEV infection induced the generation of superoxide anion and nitric oxide in rat cortical glial cells. Manganese superoxide dismutase, but not copper/zinc superoxide dismutase was activated by JEV infection, and this activation was blocked by pyrrolidine dithiocarbamate. In addition, the increased superoxide dismutase activity was also apparent in JEV acutely, or persistently infected continuous cell lines. These results suggest that cellular factors regulating oxidative pathway might play roles in responding to JEV infection.

Induction of nitric oxide synthase during Japanese encephalitis virus infection: evidence of protective role.

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage-derived factor (MDF) to modulate nitric oxide synthase (NOS) activity in brain and tumor necrosis factor-alpha (TNF-alpha) and the possible antiviral role of NOS during JEV infection were investigated. NOS activity and particularly that of the inducible form of NOS (iNOS) was significantly enhanced in JEV or JEV-induced MDF-treated mice. Following JEV infection, total NOS activity in brain was gradually increased from Day 3 and reached a peak on Day 6. MDF-induced NOS activity and iNOS activity were dose dependent and maximum activity was observed at 1 h after treatment. The response was sensitive to anti-MDF antibody treatment and N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS. Pretreatment of JEV-infected mice with L-NMMA increased the mortality as evident from reduced mean survival time (MST, 11.8 days) compared to placebo treated JEV-infected mice (MST, 17 days). The enhanced level of TNF-alpha observed in the early phase of JEV infection correlated well with the enhanced activity of iNOS. These observations thus provide evidence of the protective role of iNOS during JEV infection and indicate that iNOS may be a key mediator in host innate immune response to infection.

Plasma lactic dehydrogenase in mice infected with Japanese encephalitis & West Nile viruses.

A study of the total and isoenzymes fractions of lactic dehydrogenase (LDH) enzyme was carried out in the plasma of mice infected with Japanese encephalitis (JE) and West Nile (WN) viruses. Only 2 isoenzymes (LDH 4 and LDH 5) were present in control mice. There was a rapid increase of total LDH in the animals infected with the wild strain of viruses. Mice infected with wild strains of JE showed all 5 isoenzymes. LDH 1 disappeared after 12 days of infection but LDH 2 and LDH 3 persisted for 3 wk. The attenuated strain of JE did not produce any change either of the total content of the enzyme or of the isoenzyme pattern. In the plasma of mice infected with wild strains of WN viruses only 4 isoenzyme bands (LDH 2, 3, 4, 5) were detected. The LDH 3 persisted longer than LDH 2. With one strain of WN virus an extra band near LDH 4 was seen.