Screening a neurotransmitter-receptor-related inhibitor library identifies clomipramine HCl as a potential antiviral compound against Japanese encephalitis virus.

Background: Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide. JEV exhibits significant neuroinvasiveness and neurotoxicity, resulting in considerable damage to the nervous system. Japanese encephalitis is associated with high morbidity and mortality rate, seriously harming both human health and livestock production. The current lack of specific antiviral drugs means that the development of new therapeutic agents for JEV has become urgent. Methods: Anti-JEV drugs were screened from 111 inhibitors of neurotransmitter receptor-related molecules by high content technology. The antiviral effects of clomipramine HCl were evaluated through plaque assay, real-time quantitative PCR, immunofluorescence assay and western blotting assay. Bioinformatic tools were utilized to cluster the altered signaling pathway members after clomipramine HCl treatment. Finally, the anti-JEV mechanism was deeply resolved in vivo via such molecular biology and virological detection techniques. Results: In this study, we screened nine compounds with significant anti-JEV activity, of which clomipramine HCl demonstrated the most potent antiviral effect and exhibited dose-dependent activity. Mechanistically, clomipramine HCl may activate endoplasmic reticulum stress and modulate the unfolded protein response, thus inhibiting the assembly stage of JEV infection. Conclusion: This study highlights the importance of clomipramine HCl as a promising approach for JEV infection protection, which may lead to new host-directed antiviral approaches to such mosquito-borne viruses.

Exploring Embodied Intelligence in Soft Robotics: A Review.

Soft robotics is closely related to embodied intelligence in the joint exploration of the means to achieve more natural and effective robotic behaviors via physical forms and intelligent interactions. Embodied intelligence emphasizes that intelligence is affected by the synergy of the brain, body, and environment, focusing on the interaction between agents and the environment. Under this framework, the design and control strategies of soft robotics depend on their physical forms and material properties, as well as algorithms and data processing, which enable them to interact with the environment in a natural and adaptable manner. At present, embodied intelligence has comprehensively integrated related research results on the evolution, learning, perception, decision making in the field of intelligent algorithms, as well as on the behaviors and controls in the field of robotics. From this perspective, the relevant branches of the embodied intelligence in the context of soft robotics were studied, covering the computation of embodied morphology; the evolution of embodied AI; and the perception, control, and decision making of soft robotics. Moreover, on this basis, important research progress was summarized, and related scientific problems were discussed. This study can provide a reference for the research of embodied intelligence in the context of soft robotics.

Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation.

Ferroptosis is a newly discovered prototype of programmed cell death (PCD) driven by iron-dependent phospholipid peroxidation â€‹accumulation, and it has been linked to numerous organ injuries and degenerative pathologies. Although studies have shown that a variety of cell death processes contribute to JEV-induced neuroinflammation and neuronal injury, there is currently limited research on the specific involvement of ferroptosis. In this study, we explored the neuronal ferroptosis induced by JEV infection in vitro and in vivo. Our results indicated that JEV infection induces neuronal ferroptosis through inhibiting the function of the antioxidant system mediated by glutathione (GSH)/glutathione peroxidase 4 (GPX4), as well as by promoting lipid peroxidation mediated by yes-associated protein 1 (YAP1)/long-chain acyl-CoA synthetase 4 (ACSL4). Further analyses revealed that JEV E and prM proteins function as agonists, inducing ferroptosis. Moreover, we found that treatment with a ferroptosis inhibitor in JEV-infected mice reduces the viral titers and inflammation in the mouse brains, ultimately improving the survival rate of infected mice. In conclusion, our study unveils a critical role of ferroptosis in the pathogenesis of JEV, providing new ideas for the prevention and treatment of viral encephalitis.

Location of the AICA influences the severity but not occurrence of ISSNHL: A reappraisal using high-resolution 3 T MRI.

Objective: To investigate the potential influence of anatomical variation in the anterior inferior cerebellar artery (AICA) on the occurrence and severity of idiopathic sudden sensorineural hearing loss (ISSNHL). Methods: Ninety ISSNHL patients were enrolled. The anatomical location of the AICA was exhibited using high-resolution magnetic resonance imaging (MRI), and the various AICA types classified by previously reported Chavda and Gorrie methods were analyzed. The severity of hearing loss in the ipsilateral ear among different AICA types was compared. Results: Approximately 85.6% of subjects had unilateral ISSNHL (uISSNHL), and the others had bilateral ISSNHL (bISSNHL). In the uISSNHL group, the ratios of different AICA types were similar between the ipsilateral and contralateral ears. The ratios of the different AICA types in the bISSNHL group were similar to those in the uISSNHL group. In the uISSNHL group, pure tone audiometry (PTA) thresholds at 2 kHz, 4 kHz and 8 kHz of patients with Chavda type II AICA were higher than those of patients with Chavda type I and type III, with a significant difference at 4 kHz between type I and type II. There was a tendency of the PTA threshold in patients with Chavda type II or Gorrie type C to gradually increase from low to high frequency zones. Conclusion: When the AICA enters the IAC (Chavda type II) or crosses between the 7th and 8th cranial nerves (Gorrie type C), the severity and frequency of hearing impairment in ISSNHL but not the occurrence of ISSNHL will be affected.

H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection.

Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.

Photoacoustic spectroscopy-based ppb-level multi-gas sensor using symmetric multi-resonant cavity photoacoustic cell.

In this paper, we propose and experimentally demonstrate a symmetric multi-resonant cavity photoacoustic cell (MR-PAC) with dual microphones detection, based on multi-resonator photoacoustic spectroscopy (MR-PAS). The designed photoacoustic cell contains three interconnected acoustic resonators to facilitate simultaneous control of three lasers for multi-gas sensing. Two microphones are symmetrically located at both sides of photoacoustic cell to implement two-point detection. The length of acoustic resonator is about 50 mm to minimize the photoacoustic cell, and the resonant frequency is around 3000 Hz. Feasibility and performance of the MR-PAC was demonstrated by simultaneous detection of C2H2, NO and CF4 using a near infrared diode laser and two mid infrared quantum cascade lasers. The minimum detection limits (MDLs) of C2H2, NO and CF4 are 480 ppb, 260 ppb and 0.57 ppb respectively with a 1 s integration time at normal atmospheric pressure. This minimized MR-PAS system is promising for the portable multi-gas sensing.

Multiple genetic variants involved in both autoimmunity and autoinflammation detected in Chinese patients with sporadic Meniere's disease: a preliminary study.

Background: The mechanisms of Meniere's disease (MD) remain largely unknown. The purpose of this study was to identify possible genetic variants associated with immune regulation in MD. Methods: The whole immune genome of 16 Chinese patients diagnosed with sporadic MD was sequenced using next-generation sequencing. Results: Definite pathological variants of MEFV (c.1223G>A, c.1105C>T), COL7A1 (c.5287C>T), and ADA (c.445C>T) contributing to the clinical phenotype were found in three patients. Limited and likely pathological variants of TLR3 (c.2228G>A) and RAB27A (c.560G>A) were detected in one patient each. The following definite pathological variants impairing the structure and function of translated proteins were detected in 10 patients, and multigene variants occurred in five patients: PRF1 (c.710C>A), UNC13D (c.1228A>C), COLEC11 (c.169C>T), RAG2 (c.200G>C), BLM (c.1937G>T), RNF31 (c.2533G>A), FAT4 (c.11498A>G), PEPD (c.788A>G), TNFSF12 (c.470G>A), VPS13B (c.11972A>T), TNFRSF13B (c.226G>A), ERCC6L2 (c.4613A>G), TLR3 (c.2228G>A), ADA (c.445C>T), PEPD (c.151G>A), and MOGS (c.2470G>A). The following limited pathological variants impairing the structure and function of translated proteins were detected in five patients, with double gene variants identified in one patient: EXTL3 (c.1396G>A), MTHFD1 (c.2057G>A), FANCA (c.2039T>C), LPIN2 (c.1814C>T), NBAS (c.4049T>C), and FCN3 (c.734G>A). Conclusion: Patients with sporadic MD carry multiple genetic variants involved in multiple steps of immune regulation, which might render patients susceptible to developing inflammation via both autoimmune and autoinflammation mechanisms upon internal stress.

C-Band optical 90-degree hybrid using thin film lithium niobate.

The integrated optical 90-degree hybrid is a crucial component for coherent receivers. Here, we simulate and fabricate a 4 × 4 multimode interference coupler as a 90-degree hybrid using thin film lithium niobate (TFLN). The device features low loss (0.37 dB), high common mode rejection ratio (over 22 dB), compact footprint, and small phase error (below 2°) within the whole C-band experimentally, which is promising for integration with coherent modulators and photodetectors for TFLN-based high-bandwidth optical coherent transceivers.

Elevated G-CSF, IL8, and HGF in patients with definite Meniere's disease may indicate the role of NET formation in triggering autoimmunity and autoinflammation.

The etiology and mechanism causing Meniere's disease (MD) are not understood. The present study investigated the possible molecular mechanism of autoimmunity and autoinflammation associated with MD. Thirty-eight patients with definite MD and 39 normal volunteers were recruited, and 48 human cytokines/chemokines were quantified. In patients with MD pure tone audiograms, tympanograms and standard blood tests were performed. The mean hearing loss in the worse ear was 44.1 dB nHL. Compared to the referents, the concentrations of TNFα, IL1α, IL8, CTACK, MIP1α, MIP1ß, G-CSF, and HGF in the sera of patients with MD were significantly elevated, while those of TRAIL and PDGFBB were significantly decreased. The area under the receiver operating characteristic curve (AUC) showed that G-CSF, MIP1α, and IL8 were above 0.8 and could be used to diagnose MD (p < 0.01), and the AUCs of CTACK and HGF were above 0.7 and acceptable to discriminate the MD group from the control group (p < 0.01). The revised AUCs (1 - AUC) of TRAIL and PDGFBB were above 0.7 and could also be used in the diagnosis of MD (p < 0.01). The linear regression showed significant correlations between MIP1α and GCSF, between IL2Rα and GCSF, between IL8 and HGF, between MIP1α and IL8, and between SCF and CTACK; there was a marginal linear association between IP10 and MIP1α. Linear regression also showed that there were significant age-related correlations of CTACK and MIG expression in the MD group (p < 0.01, ANOVA) but not in the control group. We hypothesize that G-CSF, IL8, and HGF, which are involved in the development of neutrophil extracellular traps (NETs) and through various mechanisms influence the functions of macrophages, lymphocytes, and dendritic cells, among others, are key players in the development of EH and MD and could be useful in elucidating the pathophysiological mechanisms leading to MD. Biomarkers identified in the present study may suggest that both autoimmune and autoinflammatory mechanisms are involved in MD. In the future, it will be valuable to develop a cost-effective method to detect G-CSF, IL8, HGF, CTACK, MIP1α, TRAIL, and PDGFBB in the serum of patient that have diagnostic relevance.

Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME.

Zika virus (ZIKV) can be transmitted from mother to fetus during pregnancy, causing adverse fetal outcomes. Several studies have indicated that ZIKV can damage the fetal brain directly; however, whether the ZIKV-induced maternal placental injury contributes to adverse fetal outcomes is sparsely defined. Here, we demonstrated that ZIKV causes the pyroptosis of placental cells by activating the executor gasdermin E (GSDME) in vitro and in vivo. Mechanistically, TNF-α release is induced upon the recognition of viral genomic RNA by RIG-I, followed by activation of caspase-8 and caspase-3 to ultimately escalate the GSDME cleavage. Further analyses revealed that the ablation of GSDME or treatment with TNF-α receptor antagonist in ZIKV-infected pregnant mice attenuates placental pyroptosis, which consequently confers protection against adverse fetal outcomes. In conclusion, our study unveils a novel mechanism of ZIKV-induced adverse fetal outcomes via causing placental cell pyroptosis, which provides new clues for developing therapies for ZIKV-associated diseases.

MEFV, IRF8, ADA, PEPD, and NBAS gene variants and elevated serum cytokines in a patient with unilateral sporadic Meniere's disease and vascular congestion over the endolymphatic sac.

The etiology and underlying mechanism of Meniere's disease (MD) development are still unknown, although inflammation and autoimmunity have been implicated as underlying mechanisms. The human endolymphatic sac (ES) has been reported to have innate and adaptive immune capacity in local immune reactions. In vivo demonstration of inflammation of the ES in patients with MD is missing in the literature. We report the case of a 47-year-old female patient diagnosed with unilateral MD with genetic variants and cytokine markers indicating inflammation and vascular congestion of the ES. Endolymphatic hydrops in the right cochlea (grade 2) and vestibulum (grade 3) were detected using MRI. She carried heterozygous variants in MEFV (c.442G > C), IRF8 (c.1157G > T), ADA (c.445C > T), PEPD (c.151G > A), NBAS (c.4049T > C), CSF2RB (c.2222C > T), HPS6 (c.277G > T), IL2RB (c.1109C > T), IL12RB1 (c.1384G > T), IL17RC (c.260_271del GCAAGAGC TGGG), LIG1 (c.746G > A), RAG1 (c.650C > A), and SLX4 (c.1258G > C, c.5072A > G). In the serum, the levels of granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein 1α, and IL7 were significantly elevated, and the level of IL2Rα was reduced. Intratympanic administration of dexamethasone temporarily alleviated her hearing loss. Her vertigo was significantly relieved but remained slight after ES administration of corticosteroids.

Dexamethasone does not affect endolymphatic hydrops (EH) in patients with Meniere's disease within 24 h, and intratympanic administration of gadolinium plus dexamethasone simplifies high-quality imaging of EH using a novel protocol of 7 min.

Background: Intratympanic administration of gadolinium chelate allows for a better visualization of endolymphatic hydrops (EH) using MRI than intravenous injection and was recently further improved to facilitate high-quality imaging of EH in 7 min. The aim of the present study was to simplify the intratympanic administration protocol by mixing gadolinium chelate with therapeutic dexamethasone and to evaluate the effects of this mixture on the visualization of EH in MRI. Materials and methods: In an in vitro study, the potential impact of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) on the stability of dexamethasone was evaluated by analyzing dynamic changes in dexamethasone with high-performance liquid chromatography (HPLC) after mixing with Gd-DTPA. Ten patients with definite Meniere's disease (MD) were recruited to study the potential interference of dexamethasone on MRI visualization of EH, and 49 patients with MD were recruited to evaluate the effect of intratympanic injection of Gd-DTPA mixed with dexamethasone on MRI of EH using a 3T MR machine and a novel heavily T2-weighted 3-dimensional fluid-attenuated inversion recovery reconstructed using a magnitude plus zero-filled interpolation (hT2FLAIR-MZFI) sequence. Results: The retention times and peak area of dexamethasone in HPLC were not modified by the addition of Gd-DTPA. EH grading in the cochlea and vestibule was not influenced in any ear by intratympanic injection of dexamethasone. Excellent inner ear images were obtained from all patients, and EHs with various grades were displayed. There were significant correlations between diagnosis and cochlear EH (p < 0.01, Spearman's Rho), between diagnosis and vestibular EH (p < 0.01, Spearman's Rho), and between cochlear and vestibular EH (p < 0.01, Spearman's Rho). The distribution of Gd-DTPA plus dexamethasone negatively correlated with the grade of vestibular EH. Injury of the endolymph-perilymph barrier was detected in one cochlea and three vestibules of 59 inner ears with MD. Conclusions: Intratympanic administration of Gd-DTPA plus dexamethasone yielded high-quality MRI images of EH in patients with MD using a novel 7-min protocol and simplified the clinical application. Intratympanic administration of Gd-DTPA plus dexamethasone might be used to test its therapeutic effect in future work. Level of evidence: 3.

Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is an important zoonotic pathogen, which causes central nervous system symptoms in humans and reproductive disorders in swine. It has led to severe impacts on human health and the swine industry; however, there is no medicine available for treating yet. Therefore, vaccination is the best preventive measure for this disease. In the study, a modified mRNA vaccine expressing the prM and E proteins of the JEV P3 strain was manufactured, and a mouse model was used to assess its efficacy. The mRNA encoding prM and E proteins showed a high level of protein expression in vitro and were encapsulated into a lipid nanoparticle (LNP). Effective neutralizing antibodies and CD8+ T-lymphocytes-mediated immune responses were observed in vaccinated mice. Furthermore, the modified mRNA can protect mice from a lethal challenge with JEV and reduce neuroinflammation caused by JEV. This study provides a new option for the JE vaccine and lays a foundation for the subsequent development of a more efficient and safer JEV mRNA vaccine.

Nucleotide-Binding Oligomerization Domain 1 (NOD1) Positively Regulates Neuroinflammation during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that invades the central nervous system and causes neuroinflammation and extensive neuronal cell death. Nucleotide-binding oligomerization domain 1 (NOD1) is a type of pattern recognition receptor that plays a regulatory role in both bacterial and nonbacterial infections. However, the role of NOD1 in JEV-induced neuroinflammation remains undisclosed. In this study, we evaluated the effect of NOD1 activation on the progression of JEV-induced neuroinflammation using a human astrocytic cell line and NOD1 knockout mice. The results showed that JEV infection upregulated the mRNA and protein expression of NOD1, ultimately leading to an enhanced neuroinflammatory response in vivo and in vitro. Inhibition of NOD1 in cultured cells or mice significantly abrogated the inflammatory response triggered by JEV infection. Moreover, compared to the wild-type mice, the NOD1 knockout mice showed resistance to JEV infection. Mechanistically, the NOD1-mediated neuroinflammatory response was found to be associated with increased expression or activation/phosphorylation of downstream receptor-interacting protein 2 (RIPK2), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Jun N-terminal protein kinase (JNK), and NF-κB signaling molecules. Thus, NOD1 targeting could be a therapeutic approach to treat Japanese encephalitis. IMPORTANCE Neuroinflammation is the main pathological manifestation of Japanese encephalitis (JE) and the most important factor leading to morbidity and death in humans and animals infected by JEV. An in-depth understanding of the basic mechanisms of neuroinflammation will contribute to research on JE treatment. This study proved that JEV infection can activate the NOD1-RIPK2 signal cascade to induce neuroinflammation through the proven downstream MAPK, ERK, JNK, and NF-κB signal pathway. Thus, our study unveiled NOD1 as a potential target for therapeutic intervention for JE.

Screening of novel synthetic derivatives of dehydroepiandrosterone for antivirals against flaviviruses infections.

Flaviviruses are important arthropod-borne pathogens that represent an immense global health problem. Their unprecedented epidemic rate and unpredictable clinical features underscore an urgent need for antiviral interventions. Dehydroepiandrosterone (DHEA) is a natural occurring adrenal-derived steroid in the human body that has been associated in protection against various infections. In the present study, the plaque assay based primary screening was conducted on 32 synthetic derivatives of DHEA against Japanese encephalitis virus (JEV) to identify potent anti-flaviviral compounds. Based on primary screening, HAAS-AV3026 and HAAS-AV3027 were selected as hits from DHEA derivatives that exhibited strong antiviral activity against JEV (IC50 â€‹= â€‹2.13 and 1.98 â€‹µmol/L, respectively) and Zika virus (ZIKV) (IC50 â€‹= â€‹3.73 and 3.42 â€‹µmol/L, respectively). Mechanism study indicates that HAAS-AV3026 and HAAS-AV3027 do not exhibit inhibitory effect on flavivirus binding and entry process, while significantly inhibit flavivirus infection at the replication stage. Moreover, indirect immunofluorescence assay, Western blot analyses, and quantitative reverse transcription-PCR (qRT-PCR) revealed a potent antiviral activity of DHEA derivatives hits against JEV and ZIKV in terms of inhibition of viral infection, protein production, and viral RNA synthesis in Vero cells. Taken together, our results may provide a basis for the development of new antivirals against flaviviruses.

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice.

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.

Nuclear localization of Zika virus NS5 contributes to suppression of type I interferon production and response.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which caused an unprecedented epidemic in Latin America. Among all viral non-structural proteins in flavivirus, NS5 is the most highly conserved and has multiple crucial functions, including participating in viral RNA replication and suppressing host innate immunity. Although ZIKV NS5 prominently localizes in the nucleus during infection, its specific nuclear localization signal (NLS), and its role in viral replication and pathogenesis remain controversial. Here, we identified aa 11-90 and aa 370-406 regions that contain NLSs, which are critical for nuclear localization of ZIKV NS5. Further experiments demonstrated that nuclear localization of ZIKV NS5 predominantly participates in suppression of interferon regulatory factor 3 (IRF3)-mediated activation of type I IFN (IFN-I) transcription and inhibition of IFN-I downstream response independent of its effect on signal transducers and activators of transcription 2 (STAT2) degradation. These results suggest that subcellular localization of NS5 is important for its function on innate immune suppression, which provides new insight into ZIKV pathogenesis.

Decanoyl-Arg-Val-Lys-Arg-Chloromethylketone: An Antiviral Compound That Acts against Flaviviruses through the Inhibition of Furin-Mediated prM Cleavage.

Flaviviruses, such as Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and West Nile virus (WNV), are important arthropod-borne pathogens that present an immense global health problem. Their unpredictable disease severity, unusual clinical features, and severe neurological manifestations underscore an urgent need for antiviral interventions. Furin, a host proprotein convertase, is a key contender in processing flavivirus prM protein to M protein, turning the inert virus to an infectious particle. For this reason, the current study was planned to evaluate the antiviral activity of decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a specific furin inhibitor, against flaviviruses, including ZIKV and JEV. Analysis of viral proteins revealed a significant increase in the prM/E index of ZIKV or JEV in dec-RVKR-cmk-treated Vero cells compared to DMSO-treated control cells, indicating dec-RVKR-cmk inhibits prM cleavage. Plaque assay, qRT-PCR, and immunofluorescence assay revealed a strong antiviral activity of dec-RVKR-cmk against ZIKV and JEV in terms of the reduction in virus progeny titer and in viral RNA and protein production in both mammalian cells and mosquito cells. Time-of-drug addition assay revealed that the maximum reduction of virus titer was observed in post-infection treatment. Furthermore, our results showed that dec-RVKR-cmk exerts its inhibitory action on the virus release and next round infectivity but not on viral RNA replication. Taken together, our study highlights an interesting antiviral activity of dec-RVKR-cmk against flaviviruses.

Japanese Encephalitis Virus NS1' Protein Antagonizes Interferon Beta Production.

Japanese encephalitis virus (JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a 52-amino acid C-terminal extension of NS1, is generated with a -1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected (rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus (pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type I interferon (IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-ß and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.

p21-Activated Kinase 4 Signaling Promotes Japanese Encephalitis Virus-Mediated Inflammation in Astrocytes.

Japanese encephalitis virus (JEV) targets central nervous system, resulting in neuroinflammation with typical features of neuronal death along with hyper activation of glial cells. Exploring the mechanisms responsible for the JEV-caused inflammatory response remains a pivotal area of research. In the present study, we have explored the function of p21-activated kinase 4 (PAK4) in JEV-mediated inflammatory response in human astrocytes. The results showed that JEV infection enhances the phosphorylation of PAK4 in U251 cells and mouse brain. Knockdown of PAK4 resulted in decreased expression of inflammatory cytokines that include tumor necrosis factor alpha, interleukin-6, interleukin-1ß, and chemokine (C-C motif) ligand 5 and interferon ß upon JEV infection, suggesting that PAK4 signaling promotes JEV-mediated inflammation. In addition, we found that knockdown of PAK4 led to the inhibition of MAPK signaling including ERK, p38 MAPK and JNK, and also resulted in the reduced nuclear translocation of NF-κB and phosphorylation of AP-1. These results demonstrate that PAK4 signaling actively promotes JEV-mediated inflammation in human astrocytes via MAPK-NF-κB/AP-1 pathway, which will provide a new insight into the molecular mechanism of the JEV-induced inflammatory response.