RESUMEN
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.
Asunto(s)
Barrera Hematoencefálica , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Ratones Noqueados , Tirosina Quinasa c-Mer , Animales , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Barrera Hematoencefálica/metabolismo , Ratones , Encefalitis Japonesa/virología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Modelos Animales de Enfermedad , Carga Viral , Citocinas/metabolismo , Encéfalo/virología , Encéfalo/patología , Ratones Endogámicos C57BLRESUMEN
Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in experimentally infected pigs, JEV introduction into an immunologically naïve pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar, and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Replicación Viral , Animales , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/fisiología , Porcinos , Encefalitis Japonesa/transmisión , Encefalitis Japonesa/virología , Encefalitis Japonesa/veterinaria , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/transmisión , Pase Seriado , Aptitud Genética , Adaptación FisiológicaRESUMEN
Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-ß expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Interferón Tipo I , Proteínas no Estructurales Virales , Replicación Viral , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Japonesa (Especie)/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Interferón Tipo I/genética , Animales , Humanos , Porcinos , Línea Celular , Células HEK293 , Encefalitis Japonesa/virología , Encefalitis Japonesa/inmunología , Interferón beta/genética , Interferón beta/metabolismo , Interferón beta/inmunología , Interacciones Huésped-PatógenoRESUMEN
Flavivirus infection capitalizes on cellular lipid metabolism to remodel the cellular intima, creating a specialized lipid environment conducive to viral replication, assembly, and release. The Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is responsible for significant morbidity and mortality in both humans and animals. Currently, there are no effective antiviral drugs available to combat JEV infection. In this study, we embarked on a quest to identify anti-JEV compounds within a lipid compound library. Our research led to the discovery of two novel compounds, isobavachalcone (IBC) and corosolic acid (CA), which exhibit dose-dependent inhibition of JEV proliferation. Time-of-addition assays indicated that IBC and CA predominantly target the late stage of the viral replication cycle. Mechanistically, JEV nonstructural proteins 1 and 2A (NS1 and NS2A) impede 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation by obstructing the liver kinase B1 (LKB1)-AMPK interaction, resulting in decreased p-AMPK expression and a consequent upsurge in lipid synthesis. In contrast, IBC and CA may stimulate AMPK by binding to its active allosteric site, thereby inhibiting lipid synthesis essential for JEV replication and ultimately curtailing viral infection. Most importantly, in vivo experiments demonstrated that IBC and CA protected mice from JEV-induced mortality, significantly reducing viral loads in the brain and mitigating histopathological alterations. Overall, IBC and CA demonstrate significant potential as effective anti-JEV agents by precisely targeting AMPK-associated signaling pathways. These findings open new therapeutic avenues for addressing infections caused by Flaviviruses. IMPORTANCE: 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.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Antivirales , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Metabolismo de los Lípidos , Replicación Viral , Animales , Metabolismo de los Lípidos/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Ratones , Antivirales/farmacología , Humanos , Encefalitis Japonesa/tratamiento farmacológico , Encefalitis Japonesa/virología , Proteínas Quinasas Activadas por AMP/metabolismo , Chalconas/farmacología , Triterpenos/farmacología , Proteínas no Estructurales Virales/metabolismo , Infecciones por Flavivirus/tratamiento farmacológico , Infecciones por Flavivirus/virología , Infecciones por Flavivirus/metabolismo , Flavivirus/efectos de los fármacos , Línea CelularRESUMEN
Pigs are the most common amplifying hosts of the Japanese encephalitis virus (JEV). In 2016, four residents on Tsushima Island who did not own pig farms were diagnosed with JE. Therefore, a serosurvey was conducted to estimate the risk and seroprevalence of JEV after the outbreak. Sera collected from 560 Tsushima Island residents between January and September 2017 were tested for neutralizing antibodies against JEV strains JaGAr01 (genotype 3) and Muar (genotype 5). Sera collected from six wild boars between June and July 2022 were tested. The seroprevalence rates of neutralizing antibodies against JaGAr01 and Muar were 38.8% and 24.6%, respectively. High anti-JEV neutralizing antibody titers of ≥320 were identified in 16 residents, including 3 younger than 6 years with prior JEV vaccination, 2 in their 40s, and 11 older than 70. However, no anti-JEV-specific IgM was detected. Residents who engaged in outdoor activities had higher anti-JEV antibody titers. Sera from wild boars were negative for JEV RNA, but four of six samples contained neutralizing antibodies against JEV. Therefore, JEV transmission continues on Tsushima Island, even in the absence of pig farms, and wild boars might serve as the amplifying hosts.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Sus scrofa , Enfermedades de los Porcinos , Animales , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/veterinaria , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/virología , Encefalitis Japonesa/inmunología , Porcinos , Sus scrofa/virología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Estudios Seroepidemiológicos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/inmunología , Humanos , Masculino , Femenino , Genotipo , Japón/epidemiologíaRESUMEN
Objective: Genotypes (G) 1, 3, and 5 of the Japanese encephalitis virus (JEV) have been isolated in China, but the dominant genotype circulating in Chinese coastal areas remains unknown. We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis (JE) in the coastal provinces of China. Methods: In this study, we collected serum specimens from patients with JE in three coastal provinces of China (Guangdong, Zhejiang, and Shandong) from 2018 to 2020 and conducted JEV cross-neutralization tests against G1, G3, and G5. Results: Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong (92 patients), Zhejiang (192 patients), and Guangdong (77 patients), China, from 2018 to 2020. Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV. Two cases were confirmed to be infected with G1 JEV, 32 with G3 JEV, and two with G5 JEV. Conclusion: G3 was the primary infection genotype among JE cases with a definite infection genotype, and the infection caused by G5 JEV was confirmed serologically in China.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Genotipo , Humanos , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/virología , China/epidemiología , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Virus de la Encefalitis Japonesa (Especie)/inmunología , Femenino , Masculino , Adulto , Persona de Mediana Edad , Adulto Joven , Adolescente , Niño , Preescolar , Anciano , Anticuerpos Antivirales/sangreRESUMEN
Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Genotipo , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Virus de la Encefalitis Japonesa (Especie)/clasificación , Encefalitis Japonesa/virología , Animales , Humanos , Ratones , República de Corea , Virulencia , Ensayo de Placa Viral , Sustitución de Aminoácidos , Femenino , Mutación , Línea Celular , Ratones Endogámicos BALB C , Replicación ViralRESUMEN
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.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Técnicas de Amplificación de Ácido Nucleico , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Virus de la Encefalitis Japonesa (Especie)/genética , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Encefalitis Japonesa/diagnóstico , Encefalitis Japonesa/virología , Técnicas de Diagnóstico Molecular/métodos , Porcinos , Sistemas CRISPR-Cas , Sensibilidad y Especificidad , ARN Viral/genética , ARN Viral/análisisRESUMEN
Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Nucleolina , Fosfoproteínas , Proteínas de Unión al ARN , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células HeLa , Unión Proteica , Encefalitis Japonesa/virología , Encefalitis Japonesa/metabolismo , Interacciones Huésped-Patógeno , G-Cuádruplex , AnimalesRESUMEN
BACKGROUND: The causative agents of Acute Encephalitis Syndrome remain unknown in 68-75% of the cases. In Nepal, the cases are tested only for Japanese encephalitis, which constitutes only about 15% of the cases. However, there could be several organisms, including vaccine-preventable etiologies that cause acute encephalitis, when identified could direct public health efforts for prevention, including addressing gaps in vaccine coverage. OBJECTIVES: This study employs metagenomic next-generation-sequencing in the investigation of underlying causative etiologies contributing to acute encephalitis syndrome in Nepal. METHODS: In this study, we investigated 90, Japanese-encephalitis-negative, banked cerebrospinal fluid samples that were collected as part of a national surveillance network in 2016 and 2017. Randomization was done to include three age groups (< 5-years; 5-14-years; >15-years). Only some metadata (age and gender) were available. The investigation was performed in two batches which included total nucleic-acid extraction, followed by individual library preparation (DNA and RNA) and sequencing on Illumina iSeq100. The genomic data were interpreted using Chan Zuckerberg-ID and confirmed with polymerase-chain-reaction. RESULTS: Human-alphaherpes-virus 2 and Enterovirus-B were seen in two samples. These hits were confirmed by qPCR and semi-nested PCR respectively. Most of the other samples were marred by low abundance of pathogen, possible freeze-thaw cycles, lack of process controls and associated clinical metadata. CONCLUSION: From this study, two documented causative agents were revealed through metagenomic next-generation-sequencing. Insufficiency of clinical metadata, process controls, low pathogen abundance and absence of standard procedures to collect and store samples in nucleic-acid protectants could have impeded the study and incorporated ambiguity while correlating the identified hits to infection. Therefore, there is need of standardized procedures for sample collection, inclusion of process controls and clinical metadata. Despite challenging conditions, this study highlights the usefulness of mNGS to investigate diseases with unknown etiologies and guide development of adequate clinical-management-algorithms and outbreak investigations in Nepal.
Asunto(s)
Encefalopatía Aguda Febril , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Humanos , Nepal/epidemiología , Metagenómica/métodos , Adolescente , Preescolar , Niño , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Femenino , Encefalopatía Aguda Febril/epidemiología , Encefalopatía Aguda Febril/virología , Adulto Joven , Adulto , Lactante , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/virologíaRESUMEN
Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.
Asunto(s)
Citocinas , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Microglía , Humanos , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/virología , Encefalitis Japonesa/patología , Encefalitis Japonesa/tratamiento farmacológico , Encefalitis Japonesa/terapia , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Citocinas/inmunología , Microglía/inmunología , Microglía/virología , Microglía/patología , Animales , Antivirales/uso terapéutico , Replicación Viral/inmunología , Inmunidad Innata , Inmunoterapia/métodos , Encéfalo/virología , Encéfalo/inmunología , Encéfalo/patología , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/virología , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/tratamiento farmacológicoRESUMEN
The envelope (E) protein of the Japanese encephalitis virus (JEV) is a key protein for virus infection and adsorption of host cells, which determines the virulence of the virus and regulates the intensity of inflammatory response. The mutation of multiple aa residues in the E protein plays a critical role in the attenuated strain of JEV. This study demonstrated that the Asp to Gly, Ser, and His mutation of the E389 site, respectively, the replication ability of the viruses in cells was significantly reduced, and the viral neuroinvasiveness was attenuated to different degrees. Among them, the mutation at E389 site enhanced the E protein flexibility contributed to the attenuation of neuroinvasiveness. In contrast, less flexibility of E protein enhanced the neuroinvasiveness of the strain. Our results indicate that the mechanism of attenuation of E389 aa mutation attenuates neuroinvasiveness is related to increased flexibility of the E protein. In addition, the increased flexibility of E protein enhanced the viral sensitivity to heparin inhibition in vitro, which may lead to a decrease in the viral load entering brain. These results suggest that E389 residue is a potential site affecting JEV virulence, and the flexibility of the E protein of aa at this site plays an important role in the determination of neuroinvasiveness.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Proteínas del Envoltorio Viral , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/química , Animales , Línea Celular , Virulencia , Replicación Viral , Encefalitis Japonesa/virología , Humanos , Heparina/farmacología , Sustitución de Aminoácidos , Mutación Missense , Ratones , Mutación , Factores de Virulencia/genética , Glicoproteínas de MembranaRESUMEN
Japanese encephalitis virus (JEV) is an orthoflavivirus that causes Japanese encephalitis, a mosquito-borne viral infection that primarily affects humans and animals. JEV is a major cause of encephalitis in many parts of Asia, particularly in rural and agricultural areas. In this study, we used the IFNAR1-/- mice model to investigate alterations in cytokine and apoptotic factor levels in IFNAR1-/- mice upon JEV infection. A 5-week-adult female C57BL/6 IFN-α/ß receptor knockout (IFNAR1-/-) transgenic mice were intramuscularly inoculated with several viral titers and monitored within 10 dpi. The weight changes and survival rates were evaluated during the study period. Gene expression analysis was performed using RT-qPCR, targeting genes related to specific cytokines and apoptotic factors, to identify the inflammatory factors fluctuations associated with JEV strain KBPV-VR-27 infection in IFNAR1-/- mice. The expression of cytokine genes was enhanced in IFNAR1-/- mice infected with JEV KBPV-VR-27. Notably, a significant induction of cytokines, such as IL-13, IL-17α, IFN-ß, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-ß, and IFN-γ was exhibited in the lung. In addition, among the targeted apoptotic factors, only significant induction of Bak was observed in the brain. We also found that the spleen exhibited a higher viral load compared to the brain and lungs. In conclusion, the findings of this study shed light on the varying viral loads across targeted organs, with the brain exhibiting a lower viral load but pronounced expression of targeted pro-inflammatory cytokines in IFNAR1-/- mice.
Asunto(s)
Apoptosis , Citocinas , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta , Animales , Receptor de Interferón alfa y beta/genética , Encefalitis Japonesa/virología , Encefalitis Japonesa/genética , Encefalitis Japonesa/inmunología , Citocinas/metabolismo , Citocinas/genética , Virus de la Encefalitis Japonesa (Especie)/genética , Ratones , Femenino , Ratones Transgénicos , Modelos Animales de Enfermedad , Encéfalo/virología , InflamaciónRESUMEN
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.
Asunto(s)
Encefalopatía Aguda Febril , Humanos , India/epidemiología , Masculino , Adolescente , Femenino , Preescolar , Niño , Adulto Joven , Adulto , Encefalopatía Aguda Febril/epidemiología , Encefalopatía Aguda Febril/diagnóstico , Encefalopatía Aguda Febril/etiología , Encefalopatía Aguda Febril/virología , Lactante , Incidencia , Persona de Mediana Edad , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/diagnóstico , Encefalitis Japonesa/virología , Virus de la Encefalitis Japonesa (Especie)/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Anciano , Tifus por Ácaros/epidemiología , Tifus por Ácaros/diagnóstico , Tifus por Ácaros/microbiologíaRESUMEN
Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop lifelong neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pretreated with SCFA cocktail before JEV infection. Cytokine bead analysis, immunoblotting, and PCR were performed to analyze relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq, and bioinformatics tools were used for differentially expressed (DE) miRNAs and weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in monocyte chemoattractant protein (MCP1) and tumor necrosis factor alpha (TNFα) along with reduced expression of phospho-nuclear factor kappa B (phospho-NF-κB) was observed in SCFA conditions. Significant attenuation of histone deacetylase activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA + JEV-treated cells at 6 h post-infection. WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR-200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκßα that possibly dampened NF-κB signal activation downstream. IMPORTANCE: The gut-brain axis plays a pivotal role in the physiological state of an organism. Gut microbiota-derived metabolites are known to play a role in brain disorders including neuroviral infections. Short-chain fatty acids (SCFAs) appear to quench inflammatory markers in Japanese encephalitis virus-infected microglial cells in vitro. Mechanistically, we demonstrate the interaction between miR-200a-3p and ZBTB20 in regulating the canonical nuclear factor kappa B (NF-κB) signaling pathway via transcriptional regulation of Iκßα. Findings of this study pave the way to a better understanding of SCFA mechanisms that can be used to develop strategies against viral neuroinflammation.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Ácidos Grasos Volátiles , Inflamación , MicroARNs , Microglía , MicroARNs/genética , MicroARNs/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/genética , Microglía/virología , Microglía/metabolismo , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/farmacología , Ratones , Animales , Inflamación/genética , Inflamación/virología , Encefalitis Japonesa/virología , Encefalitis Japonesa/genética , Encefalitis Japonesa/metabolismo , Línea Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal , HumanosRESUMEN
Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.
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Autofagia , Regulación hacia Abajo , Virus de la Encefalitis Japonesa (Especie) , Proteína 2 de la Membrana Asociada a los Lisosomas , Lisosomas , Lisosomas/metabolismo , Animales , Virus de la Encefalitis Japonesa (Especie)/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Porcinos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Encefalitis Japonesa/virología , Encefalitis Japonesa/veterinaria , Línea Celular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genéticaRESUMEN
BACKGROUND: Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe. METHODS: This study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding. RESULTS: The study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV. CONCLUSIONS: Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes.
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Culex , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Mosquitos Vectores , Animales , Femenino , Culex/virología , Culex/fisiología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Encefalitis Japonesa/transmisión , Encefalitis Japonesa/virología , Mosquitos Vectores/virología , Saliva/virología , SueciaRESUMEN
Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.
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Anticuerpos Antivirales , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Inmunoglobulina M , Nanopartículas del Metal , Enfermedades de los Porcinos , Animales , Encefalitis Japonesa/veterinaria , Encefalitis Japonesa/diagnóstico , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/virología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Porcinos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Nanopartículas del Metal/química , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/sangre , Proteínas no Estructurales Virales/inmunología , Sensibilidad y Especificidad , Cromatografía de Afinidad/métodos , Oro/química , Tiras Reactivas , Reproducibilidad de los Resultados , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , HumanosRESUMEN
Culex gelidus (Diptera: Culicidae), an important vector of the Japanese encephalitis virus (JEV), contributes to human viral encephalitis in many Asian countries, including Thailand. This study represents the first investigation of the demographic patterns of Cx. gelidus populations in Thailand using cytochrome c oxidase subunit I (COI) gene analysis and wing geometric morphometrics (GM). Mosquitoes were collected from 10 provinces across six regions of Thailand in 2022. Analysis of the COI sequences (n = 182) indicated high haplotype diversity (0.882) and low nucleotide diversity (0.006), with 72 haplotypes identified. The haplotype network demonstrated no profound splits among the geographic populations. Neutral tests, including Tajima's D and Fu's Fs, displayed negative values, with a significant result observed for Fu's Fs (-33.048, p < 0.05). The mismatch distribution analysis indicated that the population does not statistically deviate from a model of sudden population expansion (SSD = 0.010, p > 0.05; Rg = 0.022, p > 0.05). The estimations suggest that the Cx. gelidus population in Thailand began its expansion approximately between 459,243 and 707,011 years ago. The Mantel test showed no significant relationship between genetic and geographic distances (r = 0.048, p > 0.05). Significant phenotypic differences (based on wing shape) were observed among most populations. Additionally, in this study, we found no significant relationships between phenotypic and genetic distances (r = 0.250, p > 0.05). Understanding the genetic and morphological dynamics of Cx. gelidus is vital for developing targeted surveillance and vector control measures. This knowledge will also help to predict how future environmental changes might affect these populations, thereby informing long-term vector management strategies.
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Culex , Complejo IV de Transporte de Electrones , Mosquitos Vectores , Alas de Animales , Animales , Tailandia , Culex/genética , Culex/virología , Culex/anatomía & histología , Complejo IV de Transporte de Electrones/genética , Mosquitos Vectores/genética , Mosquitos Vectores/anatomía & histología , Mosquitos Vectores/virología , Alas de Animales/anatomía & histología , ADN Mitocondrial/genética , Variación Genética , Haplotipos , Femenino , Encefalitis Japonesa/virología , Virus de la Encefalitis Japonesa (Especie)/genética , Masculino , FilogeniaRESUMEN
Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV) infection, continues to pose significant public health challenges worldwide despite efficient vaccines. The virus is classified into five genotypes, among which genotype V (GV) was not detected for a long period after its initial isolation in 1952, until reports emerged from China and the Republic of Korea (ROK) since 2009. The characteristics of the virus are crucial in estimating its potential epidemiological impact. However, characterization of GV JEVs has so far been limited to two strains: Muar, the original isolate, and XZ0934, isolated in China. Two additional ROK GV JEV isolates, NCCP 43279 and NCCP 43413, are currently available, but their characteristics have not been explored. Our phylogenetic analysis revealed that GV virus sequences from the ROK segregate into two clades. NCCP 43279 and NCCP 43413 belong to different clades and exhibit distinct in vitro phenotypes. NCCP 43279 forms larger plaques but demonstrates inefficient propagation in cell culture compared to NCCP 43413. In vivo, NCCP 43279 induces higher morbidity and mortality in mice than NCCP 43413. Notably, NCCP 43279 shows more severe blood-brain barrier damage, suggesting superior brain invasion capabilities. Consistent with its higher virulence, NCCP 43279 displays more pronounced histopathological and immunopathological outcomes. In conclusion, our study confirms that the two ROK isolates are not only classified into different clades but also exhibit distinct in vitro and in vivo characteristics.