Japanese Encephalitis Virus Infection Research Articles

An engineered Japanese encephalitis virus mRNA-lipid nanoparticle immunization induces protective immunity in mice.

Japanese encephalitis virus (JEV) and Zika virus (ZIKV) pose a severe threat to human health. Our previous research results, as well as those of other research groups, indicated that antibodies (Abs) induced by JEV infection or JEV vaccine vaccination could enhance ZIKV infection in vitro and exacerbate the mortality of ZIKV-infected mice, vice versa, which is known as antibody-dependent enhancement (ADE). Although studies on other flaviviruses revealed that altering the amino acid residues located in the fusion loop (FL) of envelope (E) protein can reduce the level of flavivirus-cross-reactive Abs, thereby abating the ADE of heterologous flavivirus infection, it is unclear whether this strategy is equally applicable to JEV. In this study, we constructed recombinant adenoviruses and nucleotide-modified mRNA-lipid nanoparticle (LNP) encoding JEV wild-type E protein or E protein mutant (designated as Ad5-JEV-EWT and Ad5-JEV-Emut; JEV-EWT mRNA-LNP, and JEV-Emut mRNA-LNP). We evaluated the immunogenicity of these vaccine candidates in mice and the capacity of vaccine-immune mouse sera to neutralize JEV infection or mediate ADE of ZIKV infection in vitro and in vivo. Ad5-JEV-Emut or JEV-Emut mRNA-LNP immunization induced ZIKV-cross-reactive Ab response which is dramatically lower than that induced by Ad5-JEV-EWT and JEV-EWT mRNA-LNP, respectively. The levels of JEV-neutralizing Abs induced by Ad5-JEV-Emut or JEV-Emut mRNA-LNP are comparable to that induced by Ad5-JEV-EWT and JEV-EWT mRNA-LNP, respectively. The ability of Abs induced by Ad5-JEV-Emut to enhance ZIKV infection in vitro is attenuated as compared with that induced by Ad5-JEV-EWT. Moreover, JEV-Emut mRNA-LNP immunization elicited potent T cell response similar to JEV-EWT mRNA-LNP in mice. Mice immunized with each mRNA-LNP exhibited lower level of serum viral load than the mock-immunized mice post JEV challenge. Mice receiving JEV-EWT mRNA-LNP-immune mouse sera exhibited ADE post ZIKV challenge whereas passively transferred JEV-Emut mRNA-LNP-immune mouse sera did not lead to obvious ADE of ZIKV infection in recipient mice. Most importantly, maternally acquired Abs did not enhance the mortality of 1-day-old neonates born to JEV-Emut mRNA-LNP-immunized mice post ZIKV challenge. These results suggest that optimizing the FL sequence of JEV could significantly reduce the level of JEV/ZIKV-cross-reactive Abs and abrogate the ADE of ZIKV infection, providing a promising strategy to develop effective and safety JEV vaccine.

Proteome modulation triggers potent antiviral response in Japanese Encephalitis Virus infected human macrophages.

Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic virus that claims thousands of children's lives globally every year, causing neuropsychiatric sequelae. While neuronal cell pathogenesis is a terminal consequence of JEV infection, the virus hijacks macrophages during initial replication and propagation, making macrophages critical cells of host immune defense that dictate the outcomes of infection. Though a plethora of studies have been reported using various neuronal and immune cells, a global response of human macrophages to JEV infection is yet to be explored. In this study, we assessed the kinetics of global proteome dysregulation employing an in vitro JEV infection model using human monocyte-derived macrophages (THP-1). A comparative assessment of the proteome of the infected THP-1 cells revealed differential regulation of 428 proteins at 24h post-infection (hpi), which was later increased to 443 by 48h post-infection. Global gene ontology analysis of the differentially expressed proteins highlighted several critical pathways related to immune and metabolic processes that are known to play either proviral or antiviral effects during infection. Notably, several antiviral proteins, including STAT2, OAS1, MX1, MX2, RIG-I, ISG15, and ISG20, were significantly upregulated at both time points post-infection. In contrast, a considerable downregulation of BCL-2, an anti-apoptotic protein at 48hpi indicates the activation of cell death pathways. Further, gene set enrichment analysis identified the type I interferon signaling pathway as one of the top upregulated pathways following JEV infection in human macrophages. Altogether, this study demonstrates human macrophage responses to JEV infection at the proteome level for the first time, highlighting several critical and novel antiviral proteins and pathways that not only advance our understanding of anti-JEV immunity but also aid in developing strategies to control this acute global public health menace.

NMDA receptor blockade attenuates Japanese encephalitis virus infection-induced microglia activation

Neurodegeneration and neuroinflammation are key components in the pathogenesis of Japanese Encephalitis caused by Japanese Encephalitis Virus (JEV) infection. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotoxic and pro-inflammatory properties in a cell context-dependent manner. Herein, potential roles of the NMDA receptor in excitatory neurotoxicity and neuroinflammation and effects of NMDA receptor blockade against JEV pathogenesis were investigated in rat microglia, neuron/glia, neuron cultures, and C57BL/6 mice. In microglia, JEV infection induced glutamate release and activated post-receptor NMDA signaling, leading to activation of Ca2+ mobilization and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), accompanied by pro-inflammatory NF-κB and AP-1 activation and cytokine expression. Additionally, increased Dynamin-Related Protein-1 protein phosphorylation, NAPDH Oxidase-2/4 expression, free radical generation, and Endoplasmic Reticulum stress paralleled with the reactive changes of microglia after JEV infection. JEV infection-induced biochemical and molecular changes contributed to microglia reactivity and pro-inflammatory cytokine expression. NMDA receptor antagonists MK801 and memantine alleviated intracellular signaling and pro-inflammatory cytokine expression in JEV-infected microglia. JEV infection induced neuronal cell death in neuron/glia culture associated with the concurrent production of pro-inflammatory cytokines. Conditioned media of JEV-infected microglia compromised neuron viability in neuron culture. JEV infection-associated neuronal cell death was alleviated by MK801 and memantine. Activation of NMDA receptor-related inflammatory changes, microglia activation, and neurodegeneration as well as reversal effects of memantine were revealed in the brains of JEV-infected mice. The current findings highlight a crucial role of the glutamate/NMDA receptor axis in linking excitotoxicity and neuroinflammation during the course of JEV pathogenesis, and proposes the anti-inflammatory and neuroprotective potential of NMDA receptor blockade.

Alpha-synuclein expression in neurons modulates Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) poses a significant threat, particularly to children. Despite extensive research efforts, the development of effective treatments against JEV has been impeded. One of the major setbacks is a lack of comprehensive understanding of neurotropism. The study focuses on alpha-synuclein (α-syn), a neuronal protein, and aims to determine its role in JEV pathogenesis. The present study reveals that the host cell upregulates α-syn in response to JEV infection. α-syn restrains JEV propagation by modulating superoxide dismutase 1 (SOD1) expression which further blocks JEV-induced ROS generation. Endogenous α-syn silencing led to a decrease in SOD1 expression and increased viral titer. α-syn plays a crucial role in counteracting oxidative stress through SOD1, which is essential for limiting JEV replication. This study provides broader implications for antiviral strategies and their possible role in neurodegenerative diseases; however, there is still much to explore, particularly regarding α-syn aggregation kinetics in JEV infection.

Cellular NONO protein binds to the flavivirus replication complex and promotes positive-strand RNA synthesis.

A cellular protein, non-POU-domain-containing octamer binding protein (NONO), bound to the replication complex of Japanese encephalitis virus (JEV) by directly interacting with the viral 3' UTR RNA and NS3 protein. These interactions were also identified in West Nile virus (WNV) and Zika virus (ZIKV). The infection of JEV or the expression of JEV NS3 protein in cells could induce relocation of NONO protein from the nucleus to the cytoplasm. In JEV-infected cells, the NS3, NS5, and viral RNA could be concurrently detected in the immunoprecipitation by the NONO-specific antibody, suggesting that NONO could integrate into the replication complex of JEV. Further results of co-immunoprecipitation assays showed that NONO protein interacted with NS3 helicase domains 1 and 2 by its two RNA recognize motifs (RRMs). The knockdown and knockout of NONO in cells could significantly reduce the replication of JEV and ZIKV but had no effect on the replication of vesicular stomatitis virus (VSV). The effect of NONO protein on JEV proliferation occurred during the replication stage, rather than the attachment and entry stages. The level of viral positive-strand RNA in NONO knockout cells was significantly reduced than that in wild-type cells at 12-48 h post-JEV infection. However, the level of negative-strand virus RNA had no difference between NONO knockout and wild-type cells at 12-24 h post-infection. In summary, our study identified a cellular protein that bound to the replication complex of flavivirus and facilitated the synthesis of positive-strand RNA.IMPORTANCEOver half of the world's population is at risk of flaviviruses infection, posing a serious global health concern. To date, there are no antiviral drugs or treatments for the severe symptoms caused by the infection of flaviviruses. Some cellular proteins could participate in the replication of virus, and these cellular proteins were also ideal targets in antiviral strategy. Here, we identified cellular NONO protein was recruited by flavivirus NS3 protein to the cytoplasm, serving as a "scaffold" for viral replication complex. Our findings also revealed that NONO protein was critical for flavivirus positive-strand RNA synthesis. Specific areas where NONO interacted with flavivirus NS3 proteins and viral UTRs have also been identified. These results propose a new mechanism for cellular protein to participate in flavivirus replication and also raise a new potential anti-flavivirus strategy.

Japanese encephalitis virus infection induces mitochondrial-mediated apoptosis through the proapoptotic protein BAX.

The Japanese encephalitis virus (JEV), a zoonotic flavivirus, is Asia's primary cause of viral encephalitis. JEV induces apoptosis in a variety of cells; however, the precise mechanisms underlying this apoptosis resulting from JEV infection remain to be elucidated. Our previous studies showed that the proapoptosis gene BAX may have a role in JEV proliferation. In this study, we constructed a PK-15 cell line (BAX.KO) with a knockout of the BAX gene using CRISPR/Cas9. The knockout of the BAX gene effectively inhibited the proliferation of JEV, resulting in a 39.9% decrease in viral protein levels, while BAX overexpression produced the opposite effect. We confirmed that JEV induces apoptosis of PK-15 using 4',6-diamidino-2-phenylindole (DAPI) staining and Annexin V-FITC/PI staining. Furthermore, we found that the phosphorylation of P53 and the expression levels of BAX, NOXA, PUMA, and cleaved-caspase-3/9 were significantly upregulated after JEV infection. Moreover, we found that JEV infection not only caused mitochondrial damage, the release of mitochondrial cytochrome C (Cyt C), and the downregulation of the apoptosis-inhibiting protein BCL-2 but also reduced the mitochondrial membrane potential (MOMP) and the accumulation of intracellular reactive oxygen species (ROS). These factors collectively encourage the activation of the mitochondrial apoptosis pathway. In contrast, BAX gene knockout significantly reduces the apoptotic changes caused by JEV infection. Treatment with the caspase3 inhibitor attenuated JEV-induced viral proliferation and release, leading to a decrease in viral protein levels of 46% in PK-15 cells and 30% in BAX.KO cells. In conclusion, this study clarified the molecular mechanisms of JEV-induced apoptosis and provided a theoretical basis for revealing the pathogenic mechanisms of JEV infection.

Japanese encephalitis virus-induced DNA methylation contributes to blood-brain barrier permeability by modulating tight junction protein expression

Japanese encephalitis virus (JEV) is a neurotropic and neuroinvasive flavivirus causing viral encephalitis, which seriously threatens the development of animal husbandry and human health. DNA methylation is a major epigenetic modification involved in viral pathogenesis, yet how DNA methylation affects JEV infection remains unknown. Here, we show genome-wide DNA methylation profiles in the brains of JEV-infected mice compared to mock-infected mice. JEV can significantly increase the overall DNA methylation levels in JEV-infected mouse brains. A total of 14,781 differentially methylated regions associated genes (DMGs) have been identified. Subsequently, KEGG pathway analysis suggested that DNA methylation modulates the tight junction signaling pathway, which can potentially impact the permeability of the blood-brain barrier (BBB). We demonstrate that hypermethylation of the tight junction gene Afdn promoter inhibited AFDN expression and increased monolayer permeability of mouse brain microvascular endothelial (bEnd.3) cells in an in vitro transwell assay. Collectively, this study reveals that DNA methylation is increased in a murine Japanese encephalitis model and that modulation of Afdn expression promotes BBB permeability.

Experimental Infections of Pigs with Japanese Encephalitis Virus Genotype 4.

The emergence of Japanese encephalitis virus (JEV) in eastern Australia in 2022 caused extensive reproductive disease in pigs and is a threat to public health. Groups of weaned piglets were experimentally infected with the Australian outbreak strain of JEV (genotype 4). All pigs challenged at 5 weeks of age were infected after an intradermal injection of 1 × 105.5 (n = 4) or 1 × 104.5 TCID50/pig (n = 5). Intranasal instillation was less effective at this age, infecting 3/4 pigs with the same higher dose and 1/5 with the lower dose. Intradermal injection using 1 × 105.0 TCID50/pig also infected 9/9 pigs at 11 weeks of age. Infection in all cases was confirmed by qRT-PCR of blood samples, which identified a viremia peak at 3-4 days and detected JEV-specific antibodies as early as 5 days after the challenge. The detection of JEV in oral and nasal swabs and in saliva from chew ropes was less consistent. JEV was detected in the tonsils of 21/22 infected pigs and was isolated from the tonsils of 9/9 pigs sampled 19 days after the challenge at 11 weeks of age. The infected pigs showed no clinical signs other than pyrexia on Days 4-6. Histopathology consistent with JEV infection was evident in the nervous tissues of all but two pigs sampled 28 days after the challenge and was characterized by meningitis, encephalitis and gliosis throughout the brain. Serological studies showed extensive cross-reactivity between JEV and Murray Valley encephalitis virus using blocking ELISAs. However, the determination of limiting-dilution titres allowed for the identification of the infecting virus. This in vivo infection model will be useful in evaluating JEV vaccines and for comparative pathogenesis studies with other JEV genotypes.

The modulation of proteomics and antioxidant stress is involved in the effect of nitazoxanide against Japanese encephalitis virus in vitro

Japanese encephalitis virus (JEV) is a significant circulating arbovirus flavivirus and the primary cause of viral encephalitis in Asia. Previous studies have demonstrated that nitazoxanide (NTZ), an antiparasitic gastroenteritis medication classified as a thiazolide, exhibits efficacy against JEV both in vitro and in vivo. To explore the potential antiviral mechanisms, we employed Tandem Mass Tag (TMT)-based quantitative proteomics to identify differentially expressed proteins (DEPs) among three groups: Blank cell group, JEV-infected cell group, and JEV-infected cells treated with NTZ. Our analysis revealed that NTZ treatment led to the upregulation of 30 DEPs and downregulation of 54 DEPs in JEV-infected cells. Enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these DEPs are involved in various biological processes and signaling pathways, including transport, localization, response to wounding, P53 pathway activation, and fatty acid metabolism-related pathways. Moreover, we observed that the expression trend of TMX2, a protein associated with redox homeostasis, was consistent with findings from TMT-based quantitative proteomics. Further investigations into reactive oxygen species (ROS), mitochondrial membrane potential, antioxidant enzyme activity, and the KEAP1-NRF2 pathway demonstrated that NTZ effectively regulates the KEAP1-NRF2 pathway while suppressing oxidative stress induced by JEV infection. In conclusion, the proteomic data along with antioxidant stress results presented herein provide a foundational basis for further research into the molecular mechanisms and potential targets of NTZ against JEV.

Role of RNA G-Quadruplexes in the Japanese Encephalitis Virus Genome and Their Recognition as Prospective Antiviral Targets.

G-quadruplexes (GQs) have been primarily studied in the context of cancer and neurodegenerative pathologies. However, recent research has shifted focus to their existence and functional roles in viral genomes, revealing GQ-regulated key pathways in various human pathogenic viruses. While GQ structures have been reported in the genomes of emerging and re-emerging viruses, RNA viruses have been understudied compared to DNA viruses, including notable examples such as human immunodeficiency virus-1, hepatitis C virus, Ebola virus, Nipah virus, Zika virus, and SARS-CoV-2. The flavivirus family, comprising the Japanese encephalitis virus (JEV), poses a significant global threat due to recurring outbreaks yet lacks approved antivirals. In this study, we identified and characterized eight putative G-quadruplex-forming motifs within essential genes involved in genome replication, assembly, and internalization in the host cell, conserved across different JEV isolates. The formation and stability of these motifs were validated through a multitude of biophysical and cell-based assays. The interaction and binding affinity of these motifs with the known GQ-binding ligand BRACO-19 were supported by biophysical assays, confirming the capability of these motifs to form GQ structures. Notably, BRACO-19 also exerted antiviral properties through reduction of viral replication and infectious virus titers as well as inhibition of viral protein expression, as evaluated by the cell-based assays. This comprehensive molecular characterization of G-quadruplex structures within the JEV genome highlights their potential as promising antiviral targets for intervention strategies against JEV infection through GQ-specific ligands.

Dual-lock-and-key virus-mimicking nanoprobes for ultra-high accurate and sensitive imaging of viral infections in vivo

Fluorescence in situ imaging of viral infection lesions in vivo is crucial for precise diagnosis of viral diseases and evaluation of the extent of viral infection. Nevertheless, achieving highly specific and sensitive fluorescence imaging of viral infection sites in vivo has posed a persistent challenge. Here, we developed a dual-lock-and-key virus-mimicking nanoprobe that consisted of polyamide dendrimers (PAMAM) loaded internally with molecular beacons double-triggered by apurinic/apyrimidinic nucleic acid endonuclease 1 (APE1) and viral RNA (vRNA), and surface-modified with the E protein of Japanese encephalitis virus (JEV). This activatable nanoprobe generated dramatically amplified fluorescent signals stimulated by expressed vRNA and APE1 during viral infection, enabling ultrahigh specific and sensitive imaging of the lesions of JEV infection in vivo. This study provides a potential approach for accurate and sensitive detection of viral infection levels and assessment of the efficacy of antiviral drugs in vivo.

Japanese encephalitis virus: An overview.

Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by the Japanese encephalitis virus (JEV), posing a substantial threat to human health and property safety. Until now, there has been a lack of specific therapeutic options for treating JEV infections. In this review article, we provide a comprehensive discussion of JEV's characteristics, diagnostic methodologies, vaccine development efforts, and potential anti-JEV pharmaceuticals to provide insights and references that could be used to inform and enhance strategies for the prevention and control of Japanese encephalitis.

GAS6 as a potential target to alleviate neuroinflammation during Japanese encephalitis in mouse models

Viral encephalitis is characterized by inflammation of the brain parenchyma caused by a variety of viruses, among which the Japanese encephalitis (JE) virus (JEV) is a typical representative arbovirus. Neuronal death, neuroinflammation, and breakdown of the blood brain barrier (BBB) constitute vicious circles of JE progression. Currently, there is no effective therapy to prevent this damage. Growth arrest specific gene 6 (GAS6) is a secreted growth factor that binds to the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases and has been demonstrated to participate in neuroprotection and suppression of inflammation in many central nervous system (CNS) diseases which has great potential for JE intervention. In this study, we found that GAS6 expression in the brain was decreased and was reversely correlated with viral load and neuronal loss. Mice with GAS6/TAM signalling deficiency showed higher mortality and accelerated neuroinflammation during peripheral JEV infection, accompanied by BBB breakdown. GAS6 directly promoted the expression of tight junction proteins in bEnd.3 cells and strengthened BBB integrity, partly via AXL. Mice administered GAS6 were more resistant to JEV infection due to increased BBB integrity, as well as decreased viral load and neuroinflammation. Thus, targeted GAS6 delivery may represent a strategy for the prevention and treatment of JE especially in patients with impaired BBB.

The protective role of Mertk in JEV-induced encephalitis by maintaining the integrity of blood–brain barrier

Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk−/− mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk−/− mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk−/− mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response.

Host Factor Rab4b Promotes Japanese Encephalitis Virus Replication.

Although the Japanese encephalitis virus (JEV) infects various cell types, its receptor molecules are still not clearly understood. In our laboratory's prior research, Rab4b was identified as a potential host factor that facilitates JEV infection in PK15 cells, utilizing a genome-wide CRISPR/Cas9 knockout library (PK-15-GeCKO). To further explore the effect of Rab4b on JEV replication, we used the Rab4b knockout PK15 cell line using the CRISPR/Cas9 technology and overexpressing the Rab4b PK15 cell line, with IFA, RT-qPCR, and Western blot to study the effect of Rab4b on viral replication in the whole life cycle of the JEV. The results show that the knockout of Rab4b inhibited the replication of the JEV in PK15 cells, and the overexpression of Rab4b promoted the replication of the JEV in PK15 cell lines. Furthermore, we demonstrated for the first time that host factor Rab4b facilitates the adsorption, internalization, assembly, and release of the JEV, thereby promoting JEV replication. This study enriches the regulatory network between the JEV and host factors and lays the experimental foundation for further understanding of the function of the Rab4b protein.

Two novel compounds inhibit Flavivirus infection in vitro and in vivo by targeting lipid metabolism.

This study is the inaugural utilization of a lipid compound library in antiviral drug screening. Two lipid compounds, isobavachalcone (IBC) and corosolic acid (CA), emerged from the screening, exhibiting substantial inhibitory effects on the Japanese encephalitis virus (JEV) proliferation in vitro. In vivo experiments underscored their efficacy, with IBC and CA reducing viral loads in the brain and mitigating JEV-induced histopathological changes, effectively shielding mice from fatal JEV infection. Intriguingly, IBC and CA may activate 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) by binding to its active site, curtailing the synthesis of lipid substances, and thus suppressing JEV proliferation. This indicates AMPK as a potential antiviral target. Remarkably, IBC and CA demonstrated suppression of multiple viruses, including Flaviviruses (JEV and Zika virus), porcine herpesvirus (pseudorabies virus), and coronaviruses (porcine deltacoronavirus and porcine epidemic diarrhea virus), suggesting their potential as broad-spectrum antiviral agents. These findings shed new light on the potential applications of these compounds in antiviral research.

In Vitro Antiviral Activity of Rhodiola crenulata Extract against Zika Virus and Japanese Encephalitis Virus: Viral Binding and Stability.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) can cause permanent neurological damage and death, yet no approved drugs exist for these infections. Rhodiola crenulate, an herb used in traditional Chinese medicine for its antioxidation and antifatigue properties, was studied for its antiviral activity against ZIKV and JEV in vitro. The cytotoxicity of Rhodiola crenulata extract (RCE) was evaluated using the CCK-8 reagent. Antiviral effects of RCE were assessed in ZIKV-infected or JEV-infected Vero cells via quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, fluorescent focus assay (FFA), and immunofluorescence assay (IFA). The cell-free antiviral effects of RCE were evaluated using an inactivation assay. To determine the stage of the viral life cycle affected by RCE, time-of-addition, binding, and entry assays were conducted. Three bioactive constituents of RCE (salidroside, tyrosol, and gallic acid) were tested for antiviral activity. RCE exhibited dose-dependent anti-ZIKV and anti-JEV activities at non-cytotoxic concentrations, which were likely achieved by disrupting viral binding and stability. Gallic acid exhibited antiviral activity against ZIKV and JEV. Our findings indicate that RCE disrupts viral binding and stability, presenting a potential strategy to treat ZIKV and JEV infections.

Larval Competition between Aedes and Culex Mosquitoes Carries over to Higher Arboviral Infection during Their Adult Stage.

The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations.

Japanese encephalitis virus infection causes reactive oxygen species-mediated skeletal muscle damage.

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.

Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.