Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

Early and late pathogenic events of newborn mice encephalitis experimentally induced by itacaiunas and curionópolis bracorhabdoviruses infection

In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a time-course study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain.     

Japanese encephalitis virus neurotropism is dependent on the degree of neuronal maturity.

In a study on Fischer rats, all animals infected with Japanese encephalitis virus (JEV) before the age of 13 days died, but animals infected after the age of 14 days did not die, confirming the age-dependent resistance to JEV infection in the rat brain. A study of the kinetics of JEV infection in the developing rat brain disclosed that JEV antigen disappeared in a particular pattern, i.e., from the deeper layers to the upper layers of the motor cortex, which paralleled neuronal maturation in the cortex. Fifteen-day-old rats, which were resistant to JEV infection, received intracerebral transplants of neurons taken from 19-day embryos. When these animals were infected with JEV after transplantation, viral antigen was detected only in the embryonal neurons soon after transplantation. Thus, it can be concluded that the susceptibility to JEV infection in the rat brain is closely associated with neuronal immaturity.     

Kinetics of cytokine profile during intraperitoneal inoculation of Japanese encephalitis virus in BALB/c mice model

Infection with Japanese encephalitis virus (JEV) is mostly asymptomatic/subclinical in 90% of the individuals. Host immune response during subclinical JEV infection is poorly understood. We assessed iNOS, IFN-gamma, TNF-alpha, IL-10 and IL-4 production in spleen, brain and sera of intraperitoneally challenged BALB/c mice by RT-PCR and ELISA along with brain histopathology at different days post inoculation (d.p.i.). In spleen of virus infected mice, expression of all cytokines including iNOS mRNA were upregulated till 5d.p.i. followed by decline. At 5d.p.i., IL-10 expression outcompeted TNF-alpha, IFN-gamma and IL-4. However, in the virus infected mice sera, IL-4 production predominated over TNF-alpha and IL-10 at 5d.p.i. Conversely, cytokines expression and iNOS mRNA remained unchanged in the brain of virus infected mice from 1 to 7d.p.i. A significant increase in the cytokine expression was observed at 11d.p.i. (P<0.05) in virus infected mice brain, with the predominance of IL-10 along with the presence of meningeal inflammation and viral RNA by histology and RT-PCR, respectively. We report a biased pattern of cytokine production in sera, brain and spleen of mice intraperitoneally challenged with JEV. IL-10 exerts neuroprotective function during JEV and regulates deleterious effects of proinflammatory cytokines; however, its mechanism needs further investigation.     

Nuclear localization of Japanese encephalitis virus core protein enhances viral replication

Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly(42) and Pro(43) in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly(42) and Pro(43) were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.     

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs

Background

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a “one health” strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets.

Methodology/Principal Findings

A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested.

Conclusions/Significance

Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.     

Mice with different susceptibility to Japanese encephalitis virus infection show selective neutralizing antibody response and myeloid cell infectivity

Background

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes public health problems in Asian countries. Only a limited number of JEV-infected individuals show symptoms and develop severe encephalitis, indicating host-dependent susceptibilities.

Methodology/Principal Findings

C3H/HeN and DBA/2 mice, which exhibit different mortalities when infected by intraperitoneal inoculation with JEV, were used as experimental models to compare viral pathogenesis and host responses. One hundred infectious virus particles killed 95% of C3H/HeN mice whereas only 40% of DBA/2 mice died. JEV RNA was detected with similar low levels in peripheral lymphoid organs and in the sera of both mouse strains. High levels of viral and cytokine RNA were observed simultaneously in the brains of C3H/HeN and DBA/2 mice starting on days 6 and 9 post-infection, respectively. The kinetics of the cytokines in sera correlated with the viral replication in the brain. Significantly earlier and higher titers of neutralizing antibodies were detected in the DBA/2 strain. Primary embryonic fibroblasts, bone marrow-derived dendritic cells and macrophages from the two mouse strains were cultured. Fibroblasts displayed similar JEV replication abilities, whereas DBA/2-derived myeloid antigen-presenting cells had lower viral infectivity and production compared to the C3H/HeN–derived cells.

Conclusions/Significance

Mice with different susceptibilities to JEV neuroinvasion did not show changes in viral tropism and host innate immune responses prior to viral entry into the central nervous system. However, early and high neutralizing antibody responses may be crucial for preventing viral neuroinvasion and host fatality. In addition, low permissiveness of myeloid dendritic cells and macrophages to JEV infection in vitro may be elements associated with late and decreased mouse neuroinvasion.     

Influence of enriched environment on viral encephalitis outcomes: behavioral and neuropathological changes in albino Swiss mice

An enriched environment has previously been described as enhancing natural killer cell activity of recognizing and killing virally infected cells. However, the effects of environmental enrichment on behavioral changes in relation to virus clearance and the neuropathology of encephalitis have not been studied in detail. We tested the hypothesis that environmental enrichment leads to less CNS neuroinvasion and/or more rapid viral clearance in association with T cells without neuronal damage. Stereology-based estimates of activated microglia perineuronal nets and neurons in CA3 were correlated with behavioral changes in the Piry rhabdovirus model of encephalitis in the albino Swiss mouse. Two-month-old female mice maintained in impoverished (IE) or enriched environments (EE) for 3 months were behaviorally tested. After the tests, an equal volume of Piry virus (IEPy, EEPy)-infected or normal brain homogenates were nasally instilled. Eight days post-instillation (dpi), when behavioral changes became apparent, brains were fixed and processed to detect viral antigens, activated microglia, perineuronal nets, and T lymphocytes by immuno- or histochemical reactions. At 20 or 40 dpi, the remaining animals were behaviorally tested and processed for the same markers. In IEPy mice, burrowing activity decreased and recovered earlier (8-10 dpi) than open field (20-40 dpi) but remained unaltered in the EEPy group. EEPy mice presented higher T-cell infiltration, less CNS cell infection by the virus and/or faster virus clearance, less microgliosis, and less damage to the extracellular matrix than IEPy. In both EEPy and IEPy animals, CA3 neuronal number remained unaltered. The results suggest that an enriched environment promotes a more effective immune response to clear CNS virus and not at the cost of CNS damage.     

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus,which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis.Axl,a receptor tyrosine kinase from TAM family,plays crucial role in many inflammatory diseases.We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection,which we speculate is due to the anti-inflammatory effect of Axl during JE.Currently,the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet.In this study,by using Axl deficient and heterozygous control mice,we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death,extended infiltration of inflammatory cells,and enhanced production of pro-inflammatory cytokines,in comparison to control mice.Additionally,consistent with our previous report,Axl deficiency had no impact on the infection and target cell tropism of JEV in brain.Taken together,our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.     

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group. JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis. H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV. Virus replication was found to be inhibited in the brains of animals that were adoptively transferred with JEV specific CTL as revealed by immunohistological staining as well as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.     

Parvovirus minute virus of mice strain i multiplication and pathogenesis in the newborn mouse brain are restricted to proliferative areas and to migratory cerebellar young neurons.

Newborn BALB/c mice intranasally inoculated at birth with a lethal dose of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) developed motor disabilities and intention tremors with a high incidence by the day 6 postinfection (dpi). These neurological syndromes paralleled the synthesis of virus intermediate DNA replicative forms and yield of infectious particles in the brain, with kinetics that peaked by this time. The preferred virus replicative sites in the brain were established early in the infection (2 dpi) and at the onset of clinical symptoms (6 dpi) and were compared with major regions of cellular proliferative activity found after intraperitoneal injection of bromodeoxyuridine 24 h before encephalons were subjected to immunohistochemistry detection. At 2 dpi, viral capsid antigen was located in the laterodorsal thalamic and the pontine nuclei but not in the extensive proliferative regions of the mouse brain at this postnatal day. At 6 dpi, however, the neurotropism of the MVMi was highlighted by its ability to target the subventricular zone of the ventricles, the subependymal zone of the olfactory bulb, and the dentate gyrus of the hippocampus, which are the three main germinal centers of the cerebrum in mouse postbirth neurogenesis. Unexpectedly, in the cerebellum, the MVMi capsid antigen was confined exclusively to cells that have undergone mitosis and have migrated to the internal granular layer (IGL) and not to the proliferative external granular layer (EGL), which was stained with antiproliferative cell nuclear antigen antibody and is the main target in other parvovirus infections. This result implies temporal or differentiation coupling between MVMi cycle and neuroblast morphogenesis, since proliferative granules of the EGL should primarily be infected but must migrate in a virus carrier state into the IGL in order to express the capsid proteins. During migration, many cells undergo destruction, accounting for the marked hypocellularity specifically found in the IGL and the irregular alignment of Purkinje cell bodies, both consistent histopathological hallmarks of animals developing cerebellar symptoms. We conclude that MVMi impairs postmitotic neuronal migration occurring in the first postnatal week, when, through the natural respiratory route of infection, the virus titer peaks in the encephalon. The results illustrate the intimate connection between MVMi neuropathogenesis and mouse brain morphogenetic stage, underscoring the potential of parvoviruses as markers of host developmental programs.     

Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection ( p  < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.     

Subclinical brain injury caused by H5N1 influenza virus infection

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.     

Pathogenic and Genotypic Characterization of a Japanese Encephalitis Virus Isolate Associated with Reproductive Failure in an Indian Pig Herd

Background

India is endemic to Japanese encephalitis virus (JEV) and recurrent outbreaks occur mainly in rice growing areas. Pigs are considered to be the amplifying host for JEV and infection in gestating pigs results in reproductive failure. Most studies conducted on JEV infection in Indian pigs have been serological surveys and very little is known about JEV genotypes circulating in pigs. So the potential risk posed by pigs in JEV transmission and the genetic relationship between viruses circulating in pigs, mosquitoes and humans is poorly understood.

Methodology/Principal Findings

This study was conducted in pigs with a history of reproductive failure characterized by stillborn piglets with neuropathological lesions. Japanese encephalitis (JE) suspected brain specimens inoculated intracerebrally into mice and Vero cells resulted in successful isolation of JEV/SW/IVRI/395A/2014. Clinicopathological observations in infected mice, demonstration of JEV antigen in brain, and analysis of the envelope protein identified the swine isolate as being neurovirulent. Phylogenetic analysis based on prM and E gene sequences showed that it belonged to genotype III. This swine isolate was closely related to JEV associated with the 2005 outbreak in India and JaoArS982 from Japan. Phylogenetic analysis of JEV strains collected between 1956 and 2014 in India categorized the GIII viruses into different clades blurring their spatial distribution, which has been discernible in the previous century.

Conclusions/Significance

Isolation of JEV from stillborn piglets and its close genetic relationship with viruses detected at least three decades ago in humans and mosquitoes in Japan suggests that the virus may have been circulating among Indian pigs for several decades. The close similarity between the present swine isolate and those detected in humans affected in the 2005 outbreak in Uttar Pradesh, India, suggests the need for more intensive surveillance of pigs and implementation of suitable strategies to control JE in India.     

Nuclear localization of flavivirus RNA synthesis in infected cells

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.     

Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus, MHV-S.

The pathogenesis of mouse hepatitis virus (MHV-S) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. In sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at 12 hr, then in the nerve cells of the olfactory bulbs. At this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. In the posterior part of the brain and spinal cord, virus was detected on days 3 to 4 postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. In weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day 6 postinoculation, and later infection spread to the brain and spinal cord. In weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day 5 postinoculation, increasing in titer thereafter. Histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. In the liver, spleen and intestines, however, some lesions were observed only in sucklings.     

Endothelial Japanese encephalitis virus infection enhances migration and adhesion of leukocytes to brain microvascular endothelia via MEK‐dependent expression of ICAM1 and the CINC and RANTES chemokines

Currently, the underlying mechanisms and the specific cell types associated with Japanese encephalitis‐associated leukocyte trafficking are not understood. Brain microvascular endothelial cells represent a functional barrier and could play key roles in leukocyte central nervous system trafficking. We found that cultured brain microvascular endothelial cells were susceptible to Japanese encephalitis virus (JEV) infection with limited amplification. This type of JEV infection had negligible effects on cell viability and barrier integrity. Instead, JEV‐infected endothelial cells attracted more leukocytes adhesion onto surfaces and the supernatants promoted chemotaxis of leukocytes. Infection with JEV was found to elicit the elevated production of intercellular adhesion molecule‐1, cytokine‐induced neutrophil chemoattractant‐1, and regulated‐upon‐activation normal T‐cell expressed and secreted, contributing to the aforementioned leukocyte adhesion and chemotaxis. We further demonstrated that extracellular signal‐regulated kinase was a key upstream regulator which stimulated extensive endothelial gene induction by up‐regulating cytosolic phospholipase A₂, NF‐κB, and cAMP response element‐binding protein via signals involving phosphorylation. These data suggest that JEV infection could activate brain microvascular endothelial cells and modify their characteristics without compromising the barrier integrity, making them favorable for the recruitment and adhesion of circulating leukocytes, thereby together with other unidentified barrier‐disrupting mechanisms contributing to Japanese encephalitis and associated neuroinflammation.     

Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication

Background

Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties.

Methodology/Principal Findings

In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus.

Conclusions/Significance

We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.     

Japanese Encephalitis Virus NS1′ Protein Antagonizes Interferon Beta Production

Japanese encephalitis virus(JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a52-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-b 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.