Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Abrogated inflammatory response promotes neurogenesis in a murine model of Japanese encephalitis

Background

Japanese encephalitis virus (JEV) induces neuroinflammation with typical features of viral encephalitis, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. The detrimental effects of inflammation on neurogenesis have been reported in various models of acute and chronic inflammation. We investigated whether JEV-induced inflammation has similar adverse effects on neurogenesis and whether those effects can be reversed using an anti-inflammatory compound minocycline.

Methodology/Principal Findings

Here, using in vitro studies and mouse models, we observed that an acute inflammatory milieu is created in the subventricular neurogenic niche following Japanese encephalitis (JE) and a resultant impairment in neurogenesis occurs, which can be reversed with minocycline treatment. Immunohistological studies showed that proliferating cells were replenished and the population of migrating neuroblasts was restored in the niche following minocycline treatment. In vitro, we checked for the efficacy of minocycline as an anti-inflammatory compound and cytokine bead array showed that production of cyto/chemokines decreased in JEV-activated BV2 cells. Furthermore, mouse neurospheres grown in the conditioned media from JEV-activated microglia exhibit arrest in both proliferation and differentiation of the spheres compared to conditioned media from control microglia. These effects were completely reversed when conditioned media from JEV-activated and minocycline treated microglia was used.

Conclusion/Significance

This study provides conclusive evidence that JEV-activated microglia and the resultant inflammatory molecules are anti-proliferative and anti-neurogenic for NSPCs growth and development, and therefore contribute to the viral neuropathogenesis. The role of minocycline in restoring neurogenesis may implicate enhanced neuronal repair and attenuation of the neuropsychiatric sequelae in JE survivors.     

Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis

Background

Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect.

Methodology/Principal Findings

Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment.

Conclusions/Significance

Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation.     

Dynamics of Japanese Encephalitis Virus Transmission among Pigs in Northwest Bangladesh and the Potential Impact of Pig Vaccination

Background

Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.

Methodology/Principal Findings

We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.

Conclusions/Significance

The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.     

Japanese Encephalitis Virus Induce Immuno-Competency in Neural Stem/Progenitor Cells

Background

The low immunogenicity of neural stem/progenitor cells (NSPCs) coupled with negligible expression of MHC antigens has popularized their use in transplantation medicine. However, in an inflammatory environment, the NSPCs express costimulatory molecules and MHC antigens, and also exhibit certain immunomodulatory functions. Since NSPCs are the cellular targets in a number of virus infections both during postnatal and adult stages, we wanted to investigate the immunological properties of these stem cells in response to viral pathogen.

Methodology/Principal Findings

We utilized both in vivo mouse model and in vitro neurosphere model of Japanese encephalitis virus (JEV) infection for the study. The NSPCs residing in the subventricular zone of the infected brains showed prominent expression of MHC-I and costimulatory molecules CD40, CD80, and CD86. Using Flow cytometry and fluorescence microscopy, we observed increased surface expression of co-stimulatory molecule and MHC class I antigen in NSPCs upon progressive JEV infection in vitro. Moreover, significant production of pro-inflammatory cyto/chemokines was detected in JEV infected NSPCs by Cytokine Bead Array analysis. Interestingly, NSPCs were capable of providing functional costimulation to allogenic T cells and JEV infection resulted in increased proliferation of allogenic T cells, as detected by Mixed Lymphocyte reaction and CFSE experiments. We also report IL-2 production by NSPCs upon JEV infection, which possibly provides mitogenic signals to T cells and trigger their proliferation.

Conclusion/Significance

The in vivo and in vitro findings clearly indicate the development of immunogenicity in NSPCs following progressive JEV infection, in our case, JEV infection. Following a neurotropic virus infection, NSPCs possibly behave as immunogenic cells and contribute to both the innate and adaptive immune axes. The newly discovered immunological properties of NSPCs may have implications in assigning a new role of these cells as non-professional antigen presenting cells in the central nervous system.     

The spatial heterogeneity between Japanese encephalitis incidence distribution and environmental variables in Nepal

Background

To identify potential environmental drivers of Japanese Encephalitis virus (JE) transmission in Nepal, we conducted an ecological study to determine the spatial association between 2005 Nepal JE incidence, and climate, agricultural, and land-cover variables at district level.

Methods

District-level data on JE cases were examined using Local Indicators of Spatial Association (LISA) analysis to identify spatial clusters from 2004 to 2008 and 2005 data was used to fit a spatial lag regression model with climate, agriculture and land-cover variables.

Results

Prior to 2006, there was a single large cluster of JE cases located in the Far-West and Mid-West terai regions of Nepal. After 2005, the distribution of JE cases in Nepal shifted with clusters found in the central hill areas. JE incidence during the 2005 epidemic had a stronger association with May mean monthly temperature and April mean monthly total precipitation compared to mean annual temperature and precipitation. A parsimonious spatial lag regression model revealed, 1) a significant negative relationship between JE incidence and April precipitation, 2) a significant positive relationship between JE incidence and percentage of irrigated land 3) a non-significant negative relationship between JE incidence and percentage of grassland cover, and 4) a unimodal non-significant relationship between JE Incidence and pig-to-human ratio.

Conclusion

JE cases clustered in the terai prior to 2006 where it seemed to shift to the Kathmandu region in subsequent years. The spatial pattern of JE cases during the 2005 epidemic in Nepal was significantly associated with low precipitation and the percentage of irrigated land. Despite the availability of an effective vaccine, it is still important to understand environmental drivers of JEV transmission since the enzootic cycle of JEV transmission is not likely to be totally interrupted. Understanding the spatial dynamics of JE risk factors may be useful in providing important information to the Nepal immunization program.     

Ecological niche modeling to estimate the distribution of Japanese encephalitis virus in Asia

Background

Culex tritaeniorhynchus is the primary vector of Japanese encephalitis virus (JEV), a leading cause of encephalitis in Asia. JEV is transmitted in an enzootic cycle involving large wading birds as the reservoirs and swine as amplifying hosts. The development of a JEV vaccine reduced the number of JE cases in regions with comprehensive childhood vaccination programs, such as in Japan and the Republic of Korea. However, the lack of vaccine programs or insufficient coverage of populations in other endemic countries leaves many people susceptible to JEV. The aim of this study was to predict the distribution of Culex tritaeniorhynchus using ecological niche modeling.

Methods/Principal Findings

An ecological niche model was constructed using the Maxent program to map the areas with suitable environmental conditions for the Cx. tritaeniorhynchus vector. Program input consisted of environmental data (temperature, elevation, rainfall) and known locations of vector presence resulting from an extensive literature search and records from MosquitoMap. The statistically significant Maxent model of the estimated probability of Cx. tritaeniorhynchus presence showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Further, the majority of human Japanese encephalitis (JE) cases were located in regions with higher estimated probability of Cx. tritaeniorhynchus presence.

Conclusions/Significance

Our ecological niche model of the estimated probability of Cx. tritaeniorhynchus presence provides a framework for better allocation of vector control resources, particularly in locations where JEV vaccinations are unavailable. Furthermore, this model provides estimates of vector probability that could improve vector surveillance programs and JE control efforts.     

A Preliminary Randomized Double Blind Placebo-Controlled Trial of Intravenous Immunoglobulin for Japanese Encephalitis in Nepal

Background

Japanese encephalitis (JE) virus (JEV) is a mosquito-borne flavivirus found across Asia that is closely related to West Nile virus. There is no known antiviral treatment for any flavivirus. Results from in vitro studies and animal models suggest intravenous immunoglobulin (IVIG) containing virus-specific neutralizing antibody may be effective in improving outcome in viral encephalitis. IVIG’s anti-inflammatory properties may also be beneficial.

Methodology/Principal Findings

We performed a pilot feasibility randomized double-blind placebo-controlled trial of IVIG containing anti-JEV neutralizing antibody (ImmunoRel, 400mg/kg/day for 5 days) in children with suspected JE at two sites in Nepal; we also examined the effect on serum neutralizing antibody titre and cytokine profiles. 22 children were recruited, 13 of whom had confirmed JE; 11 received IVIG and 11 placebo, with no protocol violations. One child (IVIG group) died during treatment and two (placebo) subsequently following hospital discharge. Overall, there was no difference in outcome between treatment groups at discharge or follow up. Passive transfer of anti-JEV antibody was seen in JEV negative children. JEV positive children treated with IVIG had JEV-specific neutralizing antibody titres approximately 16 times higher than those treated with placebo (p=0.2), which was more than could be explained by passive transfer alone. IL-4 and IL-6 were higher in the IVIG group.

Conclusions/Significance

A trial of IVIG for JE in Nepal is feasible. IVIG may augment the development of neutralizing antibodies in JEV positive patients. IVIG appears an appealing option for JE treatment that warrants further study.

Trial Registration

ClinicalTrials.gov NCT01856205     

Characterization of HCV interactions with Toll-like receptors and RIG-I in liver cells

Background and Aim

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.

Methods

The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.

Results

HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.

Conclusion

These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.     

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.     

Mechanisms of Adhesion and Subsequent Actions of a Haematopoietic Stem Cell Line,HPC-7, in the Injured Murine Intestinal Microcirculation In Vivo

Objectives

Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment.

Methods

Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1β or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally.

Results

IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12.

Conclusion

This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential.     

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.     

Study of Human RIG-I Polymorphisms Identifies Two Variants with an Opposite Impact on the Antiviral Immune Response

Background

RIG-I is a pivotal receptor that detects numerous RNA and DNA viruses. Thus, its defectiveness may strongly impair the host antiviral immunity. Remarkably, very little information is available on RIG-I single-nucleotide polymorphisms (SNPs) presenting a functional impact on the host response.

Methodology/Principal Findings

Here, we studied all non-synonymous SNPs of RIG-I using biochemical and structural modeling approaches. We identified two important variants: (i) a frameshift mutation (P₂₂₉fs) that generates a truncated, constitutively active receptor and (ii) a serine to isoleucine mutation (S₁₈₃I), which drastically inhibits antiviral signaling and exerts a down-regulatory effect, due to unintended stable complexes of RIG-I with itself and with MAVS, a key downstream adapter protein.

Conclusions/Significance

Hence, this study characterized P₂₂₉fs and S₁₈₃I SNPs as major functional RIG-I variants and potential genetic determinants of viral susceptibility. This work also demonstrated that serine 183 is a residue that critically regulates RIG-I-induced antiviral signaling.     

NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis

Background

Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 β (IL-1β) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown.

Methodology/Principal Findings

For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1β and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines.

Conclusion/Significance

Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1β and IL-18 maturation.     

Japanese encephalitis virus replicon-based vaccine expressing enterovirus-71 epitope confers dual protection from lethal challenges

Background

To construct safer recombinant flavivirus vaccine, we exploited Japanese encephalitis virus (JEV) replicon-based platform to generate single-round infectious particles (SRIPs) that expressed heterologous neutralizing epitope SP70 derived from enterovirus-71 (EV71). Such pseudo-infectious virus particles, named SRIP-SP70, although are not genuine viable viruses, closely mimic live virus infection to elicit immune responses within one round of viral life cycle.

Results

We found that, besides gaining of full protection to thwart JEV lethal challenge, female outbred ICR mice, when were immunized with SRIP-SP70 by prime-boost protocol, could not only induce SP70-specific and IgG2a predominant antibodies but also provide their newborns certain degree of protection against EV71 lethal challenge.

Conclusions

Our results therefore exemplify that this vaccination strategy could indeed confer an immunized host a dual protective immunity against subsequent lethal challenge from JEV or EV71.     

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

Southernmost Asia Is the Source of Japanese Encephalitis Virus (Genotype 1) Diversity from which the Viruses Disperse and Evolve throughout Asia

Background

Although a previous study predicted that Japanese encephalitis virus (JEV) originated in the Malaysia/Indonesia region, the virus is known to circulate mainly on the Asian continent. However, there are no reported systematic studies that adequately define how JEV then dispersed throughout Asia.

Methodology/Principal Findings

In order to understand the mode of JEV dispersal throughout the entire Asian continent and the factors that determine the dispersal characteristics of JEV, a phylogenetic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on all available JEV E gene sequences in GenBank, plus strains recently isolated in China. Here we demonstrate for the first time that JEV lineages can be divided into four endemic cycles, comprising southern Asia, eastern coastal Asia, western Asia, and central Asia. The isolation places of the viruses in each endemic cycle were geographically independent regardless of years, vectors, and hosts of isolation. Following further analysis, we propose that the southernmost region (Thailand, Vietnam, and Yunnan Province, China) was the source of JEV transmission to the Asian continent following its emergence. Three independent transmission routes from the south to north appear to define subsequent dispersal of JEV. Analysis of JEV population dynamics further supports these concepts.

Conclusions/Significance

These results and their interpretation provide new insights into our understanding of JEV evolution and dispersal and highlight its potential for introduction into non-endemic areas.