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Skeletal muscle wasting is a clinically proven pathology associated with Japanese encephalitis virus (JEV) infection; however, underlying factors that govern skeletal muscle damage are yet to be explored. The current study aims to investigate the pathobiology of skeletal muscle damage using a mouse model of JEV infection. Our study reveals a significant increment in viral copy number in skeletal muscle post‐JEV infection, which is associated with enhanced skeletal muscle cell death. Molecular and biochemical analysis confirms NOX2‐dependent generation of reactive oxygen species, leading to autophagy flux inhibition and cell apoptosis. Along with this, an alteration in mitochondrial dynamics (change in fusion and fission process) and a decrease in the total number of mitochondria copies were found during JEV disease progression. The study represents the initial evidence of skeletal muscle damage caused by JEV and provides insights into potential avenues for therapeutic advancement. The schematic illustrates the mechanism of skeletal muscle damage during JEV infection. It demonstrates a substantial increase in viral copy numbers in skeletal muscle following JEV infection, correlating with heightened skeletal muscle cell death. Molecular and biochemical analyses emphasize the significance of NOX2‐mediated oxidative stress, inhibition of autophagy flux, and alterations in mitochondrial dynamics, ultimately leading to skeletal muscle dysfunction and cell death (Figure 5). [ABSTRACT FROM AUTHOR]

Acute encephalitis syndrome (AES) is a major public health concern in India as the aetiology remains unknown in the majority of cases with the current testing algorithm. We aimed to study the incidence of Japanese encephalitis (JE) and determine the aetiology of non‐JE AES cases to develop an evidence‐based testing algorithm. Cerebrospinal fluid (CSF) samples were tested for Japanese encephalitis virus by ELISA and polymerase chain reaction (PCR). Multiplex real‐time PCR was done for Dengue, Chikungunya, West Nile, Zika, Enterovirus, Epstein Barr Virus, Herpes Simplex Virus, Adenovirus, Cytomegalovirus, Herpesvirus 6, Parechovirus, Parvovirus B19, Varicella Zoster Virus, Scrub typhus, Rickettsia species, Leptospira, Salmonella species, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Plasmodium species and by ELISA for Mumps and Measles virus. Of the 3173 CSF samples, 461 (14.5%) were positive for JE. Of the 334 non‐JE AES cases, 66.2% viz. Scrub typhus (25.7%), Mumps (19.5%), Measles (4.2%), Parvovirus B19 (3.9%) Plasmodium (2.7%), HSV 1 and 2 (2.4%), EBV and Streptococcus pneumoniae (2.1% each), Salmonella and HHV 6 (1.2% each) were predominant. Hence, an improved surveillance system and our suggested expanded testing algorithm can improve the diagnosis of potentially treatable infectious agents of AES in India. [ABSTRACT FROM AUTHOR]

Aims: Japanese encephalitis (JE) is endemic in India. Although pigs are considered important hosts and sentinels for JE outbreaks in people, limited information is available on JE virus (JEV) surveillance in pigs. Methods and Results: We investigated the spatio‐temporal distribution of JEV seroprevalence and its association with climate variables in 4451 samples from pigs in 10 districts of eastern Uttar Pradesh, India, over 10 years from 2013 to 2022. The mean seroprevalence of IgG (2013–2022) and IgM (2017–2022) was 14% (95% CI 12.8–15.2) and 10.98% (95% CI 9.8–12.2), respectively. Throughout the region, higher seroprevalence from 2013 to 2017 was observed and was highly variable with no predictable spatio‐temporal pattern between districts. Seroprevalence of up to 60.8% in Sant Kabir Nagar in 2016 and 69.5% in Gorakhpur district in 2017 for IgG and IgM was observed, respectively. IgG seroprevalence did not increase with age. Monthly time‐series decomposition of IgG and IgM seroprevalence demonstrated annual cyclicity (3–4 peaks) with seasonality (higher, broader peaks in the summer and monsoon periods). However, most variance was due to the overall trend and the random components of the time series. Autoregressive time‐series modelling of pigs sampled from Gorakhpur was insufficiently predictive for forecasting; however, an inverse association between humidity (but not rainfall or temperature) was observed. Conclusions: Detection patterns confirm seasonal epidemic periods within year‐round endemicity in pigs in eastern Uttar Pradesh. Lack of increasing age‐associated seroprevalence indicates that JEV might not be immunizing in pigs which needs further investigation because models that inform public health interventions for JEV could be inaccurate if assuming long‐term immunity in pigs. Although pigs are considered sentinels for human outbreaks, sufficient timeliness using sero‐surveillance in pigs to inform public health interventions to prevent JEV in people will require more nuanced modelling than seroprevalence and broad climate variables alone. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In Southeast Asia, despite the use of Japanese encephalitis vaccines and vaccination coverage, Japanese encephalitis (JE) transmission is still a major public health issue. The main vectors of this virus are mosquitoes from the genus Culex, which diversity and density are important in Southeast Asia. The main vector species of Japanese encephalitis virus (JEV) in Cambodia belong to the Vishnui subgroup. However, their morphological identification solely based on the adult stage remains challenging, making their segregation and detection difficult. In order to identify and describe the distribution of the three main JEV vector species in Cambodia, namely Culex vishnui, Cx. pseudovishnui and Cx. tritaeniorhynchus, mosquito samplings were carried out throughout the country in different environments. Phylogenetic analysis of the cytochrome c oxidase subunit I (coI) gene using maximum‐likelihood tree with ultrafast bootstrap and phylogeographic analysis were performed. The three main Culex species are phylogenetically separated, and represent two distinct clades, one with Cx. tritaeniorhynchus and the second with Cx. vishnui and Cx. pseudovishnui, the latter appearing as a subgroup of Cx. vishnui. The phylogeographic analysis shows a distribution of the Vishnui subgroup on the entire Cambodian territory with an overlapped distribution areas leading to a sympatric distribution of these species. The three JEV vector species are geographically well‐defined with a strong presence of Cx. pseudovishnui in the forest. Combined with the presence of Cx. tritaeniorhynchus and Cx. vishnui in rural, peri‐urban, and urban areas, the presence of JEV‐competent vectors is widespread in Cambodia. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is the foremost cause of viral encephalitis in Southeast Asia and Australia leading to approximately 68 000 clinical cases and about 13 600−20 400 deaths annually. Vaccination is not completely sure and safe. Despite this, no specific antiviral has been available or approved for JEV infection yet and treatment is generally symptomatic. Therefore, this study aims to examine the antiviral activity of natural compounds against JEV proteins. The antiviral activity of natural compounds was investigated via molecular docking, cytopathic effect (CPE) inhibition assay, western blotting, and indirect immunofluorescence assay. Physiochemical, pharmacokinetics, and toxicity analysis were evaluated for the safety and efficacy of natural compounds. Network pharmacology‐based approaches have been used to study the molecular mechanisms of drug‐target interactions. Molecular docking results suggested that the NS5 protein of JEV is the major target for natural compounds. Network pharmacology‐based analysis revealed that these drugs majorly target IL6, AKT1, tumor necrosis factor (TNF), and PTGS2 to regulate key immune and inflammatory pathways such as nuclear factor kappa B, PI3K‐Akt, and TNF signaling, during JEV infection. Our in vitro results show that among the natural compounds, curcumin provides the highest protection against JEV infection via reducing the JEV‐induced CPE (IC50 = 5.90 ± 0.44 µM/mL), and reduces the expression of NS5 protein, IL6, AKT1, TNF‐α, and PTGS2. However, other natural compounds also provide protection to some extent but their efficacy is lower compared to curcumin. Therefore, this study shows that natural compounds, mainly curcumin, may offer novel therapeutic avenues for the treatment of JEV via inhibiting key viral proteins and regulating crucial host pathways involved in JEV replication. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) infection has been recognized as a serious disease in humans. Wildlife animal infections due to JEV have not been well described. This study identified JEV infection in two deceased meerkats in Thailand, with clinical signs of neurological disease. Histopathology of brains revealed severe lymphoplasmacytic necrotizing meningoencephalitis, while similar inflammation was observed in the lung and liver. Partial JEV sequences were identified from the formalin‐fixed paraffin‐embedded‐derived brain sections of two meerkats and were found to be genetically similar to a JEV strain detected in China but not from a local strain. Using immunohistochemistry, the virus was identified in neurons and glial cells, and also found in bronchial glands, Kupffer's cells in liver, lymphocytes in the spleen and pancreatic acini, which suggests extraneural infection. Transmission electron microscopy confirmed the presence of spheroid viral particles in the lungs. These findings may suggest that infection of extraneural organs in meerkats is similar to that described in JEV‐infected humans. In conclusion, this study identified the first JEV infection in meerkats as an interesting case study. The JEV should be considered as an important differential diagnosis in meerkats with encephalitis. Further surveillance on JEV infection in meerkats and other wildlife species in a large cohort is needed in the future study. [ABSTRACT FROM AUTHOR]

Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito‐borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV‐ and JEV‐induced neuronal death has not been well investigated. In the present study examined the differences in the mechanisms involved in virus‐induced cell death and innate immune responses between the DTMUV KPS54A61 strain and the JEV JaGAr‐01 strain using primary duck neurons (DN) and duck fibroblasts (CCL‐141). DN and CCL‐141 were permissive for the infection and replication of these two viruses, which up‐regulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase‐3‐dependent manner; however, DTMUV triggered more cell death than did JEV in both CCL‐141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. The levels of the mRNA expression of innate immunity‐related genes after DTMUV infection were generally higher than the levels after JEV infection, suggesting that DTMUV‐induced immune response in duck cells may exhibit toxic effects rather than protective effects. [ABSTRACT FROM AUTHOR]

A retrospective investigation of pig tissue samples from different classical swine fever virus (CSFV) outbreaks was undertaken employing RT‐PCR for possible coinfection with other swine viruses. Four samples from three different outbreaks were found to be coinfected with Japanese encephalitis virus (JEV). Phylogenetic analysis was done based on complete E gene sequenced from all four coinfected samples. This revealed a new introduction of a divergent subgroup of JEV genotype I in India. This is the first report of detection of coinfection of JEV and CSFV in pigs and the first incidence of JEV genotype I in pigs in India. [ABSTRACT FROM AUTHOR]

Purification of the enveloped virus poses a challenge as one must retain viral infectivity to preserve immunogenicity. The traditional process of virus purification is time‐consuming, laborious and hard to scale up. Here, a rapid, simple and extensible laboratory program for the purification of Japanese encephalitis virus (JEV) was developed by using differential centrifugation, ultrafiltration, Sepharose 4 fast flow gel chromatography, and CaptoTM Core 700 chromatography. The entire process recovered 61.64% of the original virus, and the purified virus particles maintained good activity and immunogenicity. The purification process described has potential application in large‐scale production of high‐purity JEV. [ABSTRACT FROM AUTHOR]

A total of 548 mosquitoes were collected from different animal farms located near to highly populated cities in Xinjiang and were subjected to metagenomic next‐generation sequencing (mNGS). The mNGS data demonstrated that 18,842 (XJ1 strain) and 1,077 (XJ2 strain) of Japanese encephalitis virus (JEV)‐related reads were detected in XJ1 and XJ2 mosquito samples collected from Wushi and Wensu counties of Aksu area, which accounted for 0.032% and 0.006% of the total clean reads generated from XJ1 and XJ2 samples, respectively. The Bayesian molecular phylogenetic analysis suggested that XJ1 and XJ2 strains belonged to JEV genotype III and were clustered with JEV strains isolated in China. Notably, Bayesian molecular time line phylogeny revealed that XJ1 strain shared its MRCA with JEV GSS strain about 67 YA, suggesting that XJ1 strain likely originated from linages closely related to GSS strain and spread to Xinjiang later. Overall, these findings suggest that Xinjiang was probably not free from JEV, and thus, a further surveillance of JEV is required in Xinjiang. [ABSTRACT FROM AUTHOR]

Background: Japanese encephalitis virus (JEV) is endemic to tropical areas in Asia and the Western Pacific. It can cause fatal encephalitis, although most infected individuals are asymptomatic. JEV is mainly transmitted to humans through the bite of an infected mosquito, but can also be transmitted through blood transfusion. To manage the potential risk of transfusion transmission, pathogen inactivation (PI) technologies, such as THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems, have been developed. We examined the efficacy of these two PI systems to inactivate JEV. Study Design and Methods: Japanese encephalitis virus–spiked plasma units were treated using the THERAFLEX MB‐Plasma system (visible light doses, 20, 40, 60, and 120 [standard] J/cm2) in the presence of methylene blue at approximately 0.8 μmol/L and spiked platelet concentrates (PCs) were treated using the THERAFLEX UV‐Platelets system (UVC doses, 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2). Samples were taken before the first and after each illumination dose and tested for infectivity using an immunoplaque assay. Results: Treatment of plasma with the THERAFLEX MB‐Plasma system resulted in an average of 6.59 log reduction in JEV infectivity at one‐sixth of the standard visible light dose (20 J/cm2). For PCs, treatment with the THERAFLEX UV‐Platelet system resulted in an average of 7.02 log reduction in JEV infectivity at the standard UVC dose (0.20 J/cm2). Conclusions: The THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems effectively inactivated JEV in plasma or PCs, and thus these PI technologies could be an effective option to reduce the risk of JEV transfusion transmission. [ABSTRACT FROM AUTHOR]

Summary: The purpose of this risk assessment (RA) was to qualitatively estimate the risk of emergence of the Japanese encephalitis virus (JEV) in the United States (US). We followed the framework for RA of emerging vector‐borne livestock diseases (de Vos et al. 2011), which consists of a structured questionnaire, whose answers to questions can be delivered in risk categories, descriptive statements, or yes or no type of answers, being supported by the literature. The most likely pathways of introduction of JEV identified were: (a) entry through infected vectors (by aircraft, cargo ships, tires, or wind); (b) import of infected viremic animals; (c) entry of viremic migratory birds; (d) import of infected biological materials; (e) import of infected animal products; (f) entry of infected humans; and (g) import/production of contaminated biological material (e.g., vaccines). From these pathways, the probability of introduction of JEV through infected adult mosquitoes via aircraft was considered very high and via ships/containers was deemed low to moderate. The probability of introduction via other pathways or modes of entry (vector eggs or larvae, hosts, and vaccines) was considered negligible. The probability of transmission of JEV was variable, ranging from low to high (in the presence of both competent vectors and hosts), depending on the area of introduction within the US. Lastly, the probability of establishment of JEV in the continental US was considered negligible. For that reason, we stopped the risk assessment at this point of the framework. This RA provides important information regarding the elements that contribute to the risk associated with the introduction of JEV in the US. This RA also indicates that infected mosquitoes transported in aircraft (and cargo ships) are the most likely pathway of JEV entry and therefore, mitigation strategies should be directed towards this pathway. [ABSTRACT FROM AUTHOR]

Neurotropic viruses, such as the rabies virus (RABV) and Japanese encephalitis virus (JEV), induce neuronal dysfunction and complication, causing neuronal damage. Currently, there are still no effective clinical treatments for neuronal injury caused by neurotropic viruses. Memantine, a drug capable of passing through the blood‐brain barrier, noncompetitively and reversibly binds to n‐methyl‐ d‐aspartic acid (NMDA) receptors. Memantine is used to treat Alzheimer's disease by blocking the activation of extra axonal ion channels, thus preventing neuronal degeneration by inhibiting the abnormal cytosolic Ca 2+ increase. To explore whether memantine can alleviate neurological disturbances caused by RABV and JEV, the following experiments were carried out: (1) for primary neurons cultured in vitro infected with RABV, the addition of memantine showed neuroprotection. (2) In the RABV challenge experiments, memantine had limited therapeutic effect, mildly extending the survival time of mice. In contrast, memantine significantly prolonged the survival time of mice infected with JEV, by reducing the intravascular cuff and inflammatory cell infiltration in mice. Furthermore, memantine decreases the amount of JEV virus in mice brain. Highlights: Memantine showed neuroprotection to RABV infected primary neurons.Memantine had mildly extended the survival time of mice infected with RABV.Memantine significantly prolonged the survival time of mice infected with JEV.Memantine decreases the amount of JEV virus in mice brain. [ABSTRACT FROM AUTHOR]

Abstract: Japanese Encephalitis Virus (JEV) is a flavivirus responsible for an important zoonotic, vector‐borne disease included in the OIE list. JEV is endemic in a large area of Asia. In Italy, JEV has been found in dead birds collected in 1997–2000 and in a pool of Culex pipiens mosquitoes collected in 2010. Viral ecology in the inter‐epidemic periods is not known. The aim of this study was to investigate JEV in FFPE archival samples of healthy birds collected in 1997–2000 in Tuscany (Italy) in the same area and a few months after collection of birds resulted infected by JEV. Different samples from 37 young birds and 83 adults were available. Immunohistochemistry detected JEV antigen only in bone marrow samples from 12 young healthy birds. Positive cells were morphologically referable to monocyte–macrophages lineage and were positive for anti‐CD11b in serial sections. Real‐time PCR detected JEV RNA in four of these samples. These results suggest that healthy birds can harbour JEV in bone marrow cells, while no other organs resulted infected. The role of healthy birds in JEV ecology should be better investigated. Surveillance programmes should include sampling of the most appropriate target organs. [ABSTRACT FROM AUTHOR]

JEV infection in CNS leads to the JE neuroinflammation. Children and old age individual have been reported to be more prone to JEV infection. MicroRNAs are endogenous, small non‐coding RNAs, which regulate the gene expression. These are ∼22 nucleotide long, conserved RNA sequence that binds at the 3′UTR of a target mRNA and regulate the post‐transcriptional gene expression. The role of microRNAs has been reported in several diseases like cancer, viral infection, neuro‐degeneration, diabetes etc. In the present study, the human microglial cells were infected with JEV (JaOArS982). The control and infected samples were subject to microarray profiling for microRNA expression. The microarray profile yielded differentially expressed microRNAs from JEV infected samples. The microRNA gene targets, gene ontology, annotations, and pathways were identified through various bioinformatics tools. Additionally, the pathways were mostly found common to “ubiquitin mediated proteolysis,” “cytokine signaling,” “maintenance of barrier function/cell junctions,” JAK/STAT pathway” “Toll‐like receptor signaling,” “Wnt‐signaling,” “adhesion molecules,” “apoptosis,” “endocytosis,” “vesicle mediated transport” etc. [ABSTRACT FROM AUTHOR]

ABSTRACT The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live-attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live-attenuated vaccine, whereas the titers of IgG2a induced by the live-attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus-specific IFN-γ+ CD4+ and CD8+ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus-specific IL-17+ CD4+ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL-17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live-attenuated and recombinant MVA vaccines. [ABSTRACT FROM AUTHOR]

Unusual rise of acute encephalitis syndrome cases (AES) were reported in July 2014 in the northern region of West Bengal, India. Investigations were carried out to characterize the outbreak and to identify the associated virus etiology. This observational study is based on 398 line listed AES cases, mostly (70.8%, 282/398) adults, with case fatality ratio of 28.9% (115/398). Japanese encephalitis virus infection was detected in 134 (49.4%) among 271 AES cases tested and most of them (79.1%, 106/134) were adults. The study reports a large outbreak of genotype III Japanese encephalitis among adults in northern region of West Bengal, India. J. Med. Virol. 88:2004-2011, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The 2′, 5′-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study. J. Med. Virol. 88:760-768, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (AES). The sole etiologic agent of JE is Japanese encephalitis virus (JEV). Although JE/AES cases have been regarded traditionally as a disease of children, a growing number of patients with JE/AES cases are also seen in the adult age group every year in the state of West Bengal, India in spite of vaccination. Therefore, a systematic study was performed to differentiate and characterize the clinico-pathological parameters and viral diversity among the patients of different age groups. Viral diversity was also evaluated from the JE/AES cases, depending on their disease severity. A total of 441 JE/AES cases were included in this study. By MAC-ELISA, 111 samples were found JEV IgM positive and among the IgM negative cases, 26 samples were found RT-PCR positive against JEV infection. Neck rigidity, abnormal behavior, convulsion, protein in CSF, WBC in CSF, and aspartate transaminase in blood differed significantly among the patients of pediatric-adolescent and adult group in both IgM positive and RT-PCR positive cases. Viral diversity was increased significantly in the pediatric-adolescent group compared to adult patients. Interestingly, with the rise in disease severity the viral diversity was found to be increased among the patients, irrespective of their age distribution. Based on clinico-pathological parameters and analysis of viral diversity, it can be concluded that viral diversity which occurs naturally is likely to affect disease severity, especially in the patients of pediatric-adolescent group. J. Med. Virol. 87:1258-1267, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (= Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus ( JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21 days post-infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus. [ABSTRACT FROM AUTHOR]

Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2′-O-methyltransferase (2′-O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2′-O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of in vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2′-O MTase activity in vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects on in vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2′-O MTase function for any member of the Reoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2′-O MTase activity and in turn, for virus fitness. [ABSTRACT FROM AUTHOR]

Japanese encephalitis ( JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus ( JEV), has recently changed from genotype III ( GIII) to genotype I ( GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT- PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT- PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT- PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT- PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance. [ABSTRACT FROM AUTHOR]

The potential of macaque Macaca nemestrina leonina in Thailand to be infected by endemic arboviruses was assessed. The prevalence of antibodies of three arboviruses actively circulating in Thailand was determined by Plaque Reduction Neutralization assay procedures using samples from captive colonies in Northern Thailand. Out of 38 macaques, 9 (24%) presented reacting antibodies against dengue virus, 5 (13%) against Japanese encephalitis virus, and 4 (10%) against Chikungunya virus. Our results indicate that the northern pig-tailed macaque in Thailand can be infected by these arboviruses, inferring therefore that their virus specific vectors have bitten them. Given that, northern pig-tailed macaque represents an abundant population, living in close range to human or in peridomestic setting, they could play a role as potential reservoir host for arboviruses circulating in Thailand. Am. J. Primatol. 76:97-102, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Persistence of RNA viruses is often, but not always, associated with the production of defective interfering (DI) particles. To investigate possible roles of DI particles and helper viruses in RNA virus persistence, persistent infection with Japanese encephalitis virus (JEV) was established in baby hamster kidney (BHK-21) cells. At the 6th and 7th serial undiluted passages of JEV on BHK-21 cells, viral persistence was established spontaneously with DI RNA generation. Seven cell clones exhibiting persistent infection were obtained from the initial BHK-21 cell batches exhibiting JEV persistence, and maintained for over 400 days. Most cell clones produced infectious particles (101-105 PFU/ml) continuously, expressed viral proteins, and resisted homologous superinfection. Two helper viruses, chvBS6-3 and chvBS7-1, were isolated from two of the seven cell clones, and characterized to investigate their roles in JEV persistence. While chvBS6-3 was restored to its full cytopathicity in the absence of DI RNA, chvBS7-1 exhibited almost no cytopathicity, regardless of DI RNA co-replication. Attenuation of chvBS7-1 did not appear to be due to inadequate adsorption or genome replication, but due to inefficient egress of the assembled progeny virions, suggesting altered helper virus emergence during JEV persistence in BHK-21 cells. These observations suggest that at least two mechanisms are involved in JEV persistence; a DI RNA-dependent mechanism, where DI RNA co-replication nullifies the helper virus's cytopathicity, or a DI RNA-independent mechanism, where the helper virus is self-attenuated. This study provides a useful in vitro tool for understanding the mechanisms underlying RNA virus persistent infections. J Med. Virol. 85:1990-2000, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age-dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons, the mechanisms underlying this association remain poorly understood. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non-permissive conditions. JEV infected undifferentiated CSM14.1 cells more efficiently than differentiated cells, resulting in production of more progeny virus in the former setting than in the latter. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon-β gene, along with smaller amounts of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play roles in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Because CSM14.1 cells became more resistant to JEV infection as they mature, this culture system can be used as an in vitro model for studying age-dependent resistance of neurons to JEV infection. [ABSTRACT FROM AUTHOR]

ABSTRACT An engineered bio-nanocapsule (BNC) comprising modified hepatitis B surface antigen L protein was used as a physical scaffold for envelope protein domain III (D3) of Japanese encephalitis virus (JEV). At the N terminus, the BNC contained a two-tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ-BNC). The Lys-rich ZZ moiety exposed on the surface of ZZ-BNC was used for chemical conjugation with the JEV D3 antigen, which had been expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ-BNC (ZZ-BNC:D3) augmented serum IgG response against JEV and increased protection against lethal JEV infection. The present study suggests that innocuous recombinant antigens, when loaded on the surface of ZZ-BNC, can be transformed to immunogenic antigens. [ABSTRACT FROM AUTHOR]

Encephalitis is an inflammation and swelling of the brain, which is often caused by a viral infection; it is an important cause of acute symptomatic seizures as well as subsequent epilepsy. Herein we describe the definition, epidemiology, and etiology of encephalitis as a cause of seizures. We then focus on encephalitis due to herpes simplex virus (the most common sporadic viral cause of encephalitis) and Japanese encephalitis virus (the most common epidemic viral cause). We also discuss the evidence for seizures occurring in the context of antibody-associated encephalitis, an increasingly important condition. Finally, we describe the acute and longer-term management of encephalitis-related seizures and their potential pathophysiologic mechanisms, concluding with the emerging etiologic role of human herpesvirus 6. [ABSTRACT FROM AUTHOR]

Japanese encephalitis was recently reported from individuals in the mountain districts of Nepal without travel history to Japanese encephalitis virus (JEV) endemic areas. We performed a cross-sectional study to estimate the seroprevalence of JEV in pigs and subsequently conducted a survey of farmers to identify risk factors associated with seropositivity. In July and August, 2010, 454 pig serum samples were collected and tested by competitive ELISA. Data from a 35-question survey of 109 pig owners were analysed using multivariate logistic regression. Seventy-six (16.7, 95% CI 13.6-20.4) pigs tested positive for anti-JEV antibodies, none of which had been vaccinated against JEV or sourced from JEV endemic areas. Risk factors associated with JEV seropositivity were 'summer abortion', 'wells as a water source', 'urban location', 'reported presence of mosquitoes' and 'lower elevation'. Our results suggest that JEV is likely circulating in the mountain districts of Nepal, and that locally acquired JEV should be considered a risk for residents and travellers in these areas. [ABSTRACT FROM AUTHOR]

K. T. Wong, K. Y. Ng, K. C. Ong, W. F. Ng, S. K. Shankar, A. Mahadevan, B. Radotra, I. J. Su, G. Lau, A. E. Ling, K. P. Chan, P. Macorelles, S. Vallet, M. J. Cardosa, A. Desai, V. Ravi, N. Nagata, H. Shimizu and T. Takasaki (2012) Neuropathology and Applied Neurobiology 38, 443-453 Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA Aims: To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. Methods: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. Results: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. Conclusions: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE. [ABSTRACT FROM AUTHOR]

ABSTRACT Japanese encephalitis virus (JEV) causes diseases that attack the human central nervous system. Traditionally, the quality control for JEV vaccines, in which the plaque reduction neutralization (PRN) titer is measured by the national control laboratories before the vaccine batches are marketed, has required laboratory animal testing. However, classical animal tests have inherent problems, including the very fact that animals are used, ethical issues, and the possibility of error. In this study, JEV antigen was measured in an in vitro assay to assess the feasibility of replacing in vivo assays that measure the PRN titers of JEV vaccines. We constructed a double-sandwich enzyme-linked immunosorbent assay (DS-ELISA) that could detect JEV envelope (E). Initially, monoclonal antibodies (mAbs) directed against the JEV E protein were generated and characterized. We isolated 18 mAbs against JEV E protein, and most were the IgG1 or IgG2a isotype. The mAbs (5F15 and 7D71) were selected as the most suitable mAb pair to detect JEV E protein. DS-ELISA with this pair detected as little as approximately 3 μg/mL JEV E protein and demonstrated a relationship between the amount of JEV E protein and the PRN titer. From these results, we surmise that this DS-ELISA may be useful, not only in terms of measuring the amount of JEV E protein, but also as a substitute for the PRN test for JEV vaccine evaluation. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 537-549. Abstract Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3′-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl β-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3′-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection. [ABSTRACT FROM AUTHOR]

Objective To compare two commercially available kits, Japanese Encephalitis-Dengue IgM Combo ELISA (Panbio Diagnostics) and JEV-CheX IgM capture ELISA (XCyton Diagnostics Limited), to a reference standard (Universiti Malaysia Sarawak – Venture Technologies VT ELISA). Methods Samples were obtained from 172/192 children presenting to a site in rural India with acute encephalitis syndrome. Results Using the reference VT ELISA, infection with Japanese encephalitis virus (JEV) was confirmed in 44 (26%) patients, with central nervous system infection confirmed in 27 of these; seven patients were dengue seropositive. Of the 121 remaining patients, 37 (31%) were JEV negative and 84 (69%) were JEV unknown because timing of the last sample tested was <10 day of illness or unknown. For patient classification with XCyton, using cerebrospinal fluid alone (the recommended sample), sensitivity was 77.8% (59.2–89.4) with specificity of 97.3% (90.6–99.2). For Panbio ELISA, using serum alone (the recommended sample), sensitivity was 72.5% (57.2–83.9) with specificity of 97.5% (92.8–99.1). Using all available samples for patient classification, sensitivity and specificity were 63.6% (95% CI: 48.9–76.2) and 98.4% (94.5–99.6), respectively, for XCyton ELISA and 75.0% (59.3–85.4) and 97.7% (93.3–99.2) for Panbio ELISA. Conclusion The two commercially available ELISAs had reasonable sensitivities and excellent specificities for diagnosing JEV. [ABSTRACT FROM AUTHOR]

Abstract: Astrocytes become activated in response to many CNS pathologies. The process of astrocyte activation remains rather enigmatic and results in so-called reactive gliosis, a reaction with specific structural and functional characteristics. Astrocytes play a vital role in regulating aspects of inflammation and in the homeostatic maintenance of the CNS. However, the responses of different human astroglial cell-lines in viral encephalitis mediated inflammation are not well documented. We have shown that Japanese encephalitis virus (JEV) infection causes morphological and functional changes in astrocytic cell-lines. We have demonstrated that besides reactive oxygen species (ROS) JEV infection differentially regulated the induction pattern of IL-6, IL-1β and IL-8. IP-10, MCP-1, MIG and RANTES secretions in different astroglial cell-lines. The expression of different proteins such as astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter/essential amino acid transporter-1 (GLAST/EAAT-1), glutamate transporter-1/essential amino acid transporter-2 (GLT-1/EAAT-2), Ceruloplasmin and Thioredoxin (TRX) expression level also differ in different human astrocyte cell-lines following infection. [Copyright &y& Elsevier]

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To monitor Japanese encephalitis virus (JEV) activity in endemic areas of Tamil Nadu, southern India, desiccated vector mosquitoes were screened for JEV antigen using ELISA, from 1996. A total of 133 233 specimens from eight index villages comprising 2816 pools (mainly Culex vishnui subgroup) were tested. Of these, 59 pools (2.1%) were positive for JEV antigen. Control measures were undertaken in positive villages accordingly. The average annual minimum infection rate was 0.8 at the beginning of the study and remained lower for nearly 8 years. A declining trend in JE cases was recorded. [ABSTRACT FROM AUTHOR]

Adult mosquitoes (Diptera: Culicidae) were collected in January and February 2000 from Saibai Island in the Torres Strait of northern Australia, and processed for arbovirus isolation during a period of Japanese encephalitis (JE) virus activity on nearby Badu Island. A total of 84 210 mosquitoes were processed for virus isolation, yielding six flavivirus isolates. Viruses obtained were single isolates of JE and Kokobera (KOK) and four of Kunjin (KUN). All virus isolates were from members of the Culex sitiens Weidemann subgroup, which comprised 53.1% of mosquitoes processed. Nucleotide sequencing and phylogenetic analysis of the pre-membrane region of the genome of JE isolate TS5313 indicated that it was closely related to other isolates from a sentinel pig and a pool of Cx. gelidus Theobald from Badu Island during the same period. Also molecular analyses of part of the envelope gene of KUN virus isolates showed that they were closely related to other KUN virus strains from Cape York Peninsula. The results indicate that flaviviruses are dynamic in the area, and suggest patterns of movement south from New Guinea and north from the Australian mainland. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the prevalence of antibodies to flaviviruses, particularly Japanese encephalitis (JE) in children. Methods: Virological and serological investigations were conducted into JE, along with dengue 2 (Den2) and West Nile viruses, in the Flavivirus group. The paediatric age group (up to 10 y), in the districts of Burdwan, Bankura, Midnapore and Purulia of West Bengal, India, was assessed for recent activity of these viruses, for 4 consecutive years from 1996 to 1999. In total, 2260 sera samples were collected, of which 204 were from acute fever cases; only 72 paired sera were available. There was a significantly higher incidence of fever cases in children belonging to the schedule caste and schedule tribes. These communities occupy the lowest level in the society and are closely associated with pigs. Results: No virus could be isolated from the sera collected from fever cases. The results of the serological survey showed the presence of antibodies to JE virus in only 13.3% of the contact sera, 33.3% of acute sera and 22.1% of convalescent sera investigated. Only 3.1% of the contact sera had antibodies to Den2 and no antibodies were found against West Nile virus. This indicates that the JE virus is in recent circulation in the population of these four districts, and as the majority of children lack substantial immunity to JE virus, they may be affected by an epidemic. Conclusion: Children up to 10 y of age in the four districts of West Bengal are mostly non-immune to JE virus. If an outbreak occurs, the majority of these populations may be affected. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. – the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00–08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only <10% of bloodmeals were from birds. The predominant species was Cx. annulirostris (88%), with relatively few (4.4%) bloodmeals obtained from humans. From all 12 locations sampled, the mean proportion of Cx. annulirostris fed on pigs (9.1%) was considerably lower than fed on other animals (90.9%). Highest rates of pig-fed mosquitoes (>30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia. [ABSTRACT FROM AUTHOR]