Japanese Encephalitis Virus Replication Research Articles

Retraction: p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication.

Retraction: p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication.

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.

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.

SLC25A12 inhibits Japanese encephalitis virus replication by interacting with the NS1 and enhancing the type I interferon response

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-β 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.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues

BackgroundMacrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies.MethodsTo elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection.ResultsNOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages.ConclusionIn light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

Japanese encephalitis virus NS1 and NS1' proteins induce vimentin rearrangement via the CDK1-PLK1 axis to promote viral replication.

Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus that causes severe encephalitis in humans, particularly in Asia. Despite the availability of a safe and effective vaccine, JEV infection remains a significant public health threat due to limited vaccination coverage. Understanding the interactions between JEV and host proteins is essential for developing more effective antiviral strategies. In this study, we investigated the role of vimentin, an intermediate filament protein, in JEV replication. Our findings reveal that JEV NS1 and NS1' proteins induce vimentin rearrangement, resulting in the formation of cage-like structures that envelop the viral replication factories (RFs), thus facilitating efficient viral replication. Our research highlights the importance of the interplay between the cytoskeleton and orthoflavivirus, suggesting that targeting vimentin could be a promising approach for the development of antiviral strategies to inhibit JEV propagation.

Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

CRISPR/Cas9-Mediated Knockout of the HuR Gene in U251 Cell Inhibits Japanese Encephalitis Virus Replication.

Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus-host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses.

N6-methyladenosine modification positively regulate Japanese encephalitis virus replication

N6-methyladenosine (m6A) is present in diverse viral RNA and plays important regulatory roles in virus replication and host antiviral innate immunity. However, the role of m6A in regulating JEV replication has not been investigated. Here, we show that the JEV genome contains m6A modification upon infection of mouse neuroblast cells (neuro2a). JEV infection results in a decrease in the expression of m6A writer METTL3 in mouse brain tissue. METTL3 knockdown by siRNA leads to a substantial decrease in JEV replication and the production of progeny viruses at 48 hpi. Mechanically, JEV triggered a considerable increase in the innate immune response of METTL3 knockdown neuro2a cells compared to the control cells. Our study has revealed the distinctive m6A signatures of both the virus and host in neuro2a cells infected with JEV, illustrating the positive role of m6A modification in JEV infection. Our study further enhances understanding of the role of m6A modification in Flaviviridae viruses.

Nuclear membrane protein SUN2 promotes replication of flaviviruses through modulating cytoskeleton reorganization mediated by NS1

Cytoskeleton is extensively recruited by flaviviruses for their infection. In this study, we uncovered an essential role of a nuclear membrane protein, SAD1/UNC84 domain protein 2 (SUN2) linking cytoskeleton and nucleoskeleton in the flavivirus replication. CRISPR/Cas9-mediated knockout of SUN2, but not SUN1, significantly reduces the replication of Zika virus (ZIKV), dengue virus (DENV), and Japanese encephalitis virus (JEV). In contrast, SUN2 does not affect the infection of non-flaviviridae RNA viruses. All three regions of SUN2 are required for its proviral effect. Mechanistically, SUN2 facilitates rearrangement of cytoskeleton and formation of replication organelles induced by viral infection, and hence promotes viral RNA synthesis. SUN2 is required for the interaction between cytoskeleton actin and ZIKV nonstructural protein 1 (NS1). Expression of dominant negative Nesprin-1 and Nesprin-2, which connect SUN2 to cytoskeleton proteins, alleviates the interaction between actin and NS1 and reduces viral replication levels. In a neonatal mouse infection model, SUN2 knockout dramatically alleviates the in vivo ZIKV replication and development of neuropathology. This work elucidates that recruitment of cytoskeleton proteins by flavivirus is coordinated by nuclear membrane proteins SUN2 and Nesprins, providing evidence for a link between nuclear membrane proteins and flavivirus infection.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

A fusion protein of vimentin with Fc fragment inhibits Japanese encephalitis virus replication.

Japanese encephalitis virus (JEV), a member of the Flaviviridae family and a flavivirus, is known to induce acute encephalitis. Vimentin protein has been identified as a potential receptor for JEV, engaging in interactions with the viral membrane protein. The Fc fragment, an integral constituent of immunoglobulins, plays a crucial role in antigen recognition by dendritic cells (DCs) or phagocytes, leading to subsequent antigen presentation, cytotoxicity, or phagocytosis. In this study, we fused the receptor of JEV vimentin with the Fc fragment of IgG and expressed the resulting vimentin-Fc fusion protein in Escherichia coli. Pull-down experiments demonstrated the binding ability of the vimentin-Fc fusion protein to JEV virion in vitro. Additionally, we conducted inhibition assays at the cellular level, revealing the ability of vimentin-Fc protein suppressing JEV replication, it may be a promising passive immunotherapy agent for JEV. These findings pave the way for potential therapeutic strategies against JEV.

Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells

BackgroundPromyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection.MethodsPML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-β signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays.ResultsOur results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-β signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection.ConclusionsThese findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-β and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

An RBM10 and NF-κB interacting host lncRNA promotes JEV replication and neuronal cell death.

Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1. RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-β was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

Melatonin inhibits Japanese encephalitis virus replication and neurotoxicity via calcineurin-autophagy pathways

BackgroundJapanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has no specific treatment except for supportive medical care. JEV is a neurotropic virus that affects the nervous system and triggers inflammation in the brain.MethodsMelatonin is used as a sleep-inducing agent in neurophysiology and may serve as a protective agent against neurological and neurodegenerative diseases. Herein, we investigated the effects of melatonin and the critical roles of the serine/threonine protein phosphatase calcineurin during JEV infection in SK-N-SH neuroblastoma cells.ResultsMelatonin treatment decreased JEV replication and JEV-mediated neurotoxicity. Calcineurin activity was increased by JEV infection and inhibited by melatonin treatment. Through calcineurin regulation, melatonin decreased the JEV-mediated neuroinflammatory response and attenuated JEV-induced autophagy.ConclusionsCalcineurin inactivation has a protective effect in JEV-infected neuronal cells, and melatonin is a novel resource for the development of anti-JEV agents.

M6A modification associated with YTHDF1 is involved in Japanese encephalitis virus infection

N6-methyladenosine (m6A), the most common modification in mammalian mRNA and viral RNA, regulates mRNA structure, stability, translation, and nuclear export. The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus causing severe neurologic disease in humans. To date, the role of m6A modification in JEV infection remains unclear. Herein, we aimed to determine the impact of m6A methylation modification on JEV replication in vitro and in vivo. Our results demonstrated that the overexpression of the m6A reader protein YTHDF1 in vitro significantly inhibits JEV proliferation. Additionally, YTHDF1 negatively regulates JEV proliferation in YTHDF1 knockdown cells and YTHDF1 knockout mice. MeRIP-seq analysis indicated that YTHDF1 interacts with several interferon-stimulated genes (ISGs), especially in IFIT3. Overall, our data showed that YTHDF1 played a vital role in inhibiting JEV replication. These findings bring novel insights into the specific mechanisms involved in the innate immune response to infection with JEV. They can be used in the development of novel therapeutics for controlling JEV infection.