EV71-infected Cells Research Articles

Astragaloside IV prevents enterovirus 71-induced pyroptosis through the TCF12-TXNIP-Keap1/Nrf2 axis

This study aimed to investigate the inhibitory effect of astragaloside IV (AS-IV) on enterovirus 71 (EV71)-induced pyroptosis. The results indicated that AST-IV inhibited EV71-induced pyroptosis in normal human gastric epithelial (GES-1) cells. Therefore, we investigated the mechanisms underlying pyroptosis inhibition by AST-IV. We found that EV71 inhibited the Nrf2-Keap1 pathway, whereas AST-IV treatment activated it. Overexpression of Nrf2 led to the inhibition of EV71-induced pyroptosis. EV71 infection and AST-IV treatment upregulated and downregulated thioredoxin interacting protein (TXNIP), respectively. Downregulation of TXNIP activated the Keap1/Nrf2 signaling pathway and inhibited EV71-induced pyroptosis. Furthermore, transcription factor 12 (TCF12) reduced the protein and mRNA levels of TXNIP, and AST-IV treatment upregulated TCF12. These results demonstrate that the TCF12-TXNIP-Keap1/Nrf2 axis participates in the inhibition of pyroptosis by AST-IV in EV71-infected GES-1 cells, suggesting that AST-IV could be used as a dietary supplement to alleviate EV71-induced pyroptosis.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

High mobility group box 1 knockdown inhibits EV71 replication and attenuates cell pyroptosis through TLR4/NF-κB/NLRP3 axis.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) in children. Nowadays, there are still no effective antiviral drugs for EV71 infection. High mobility group box 1 (HMGB1) is reported to be highly expressed in HFMD patients. However, therole and underlying mechanism of HMGB1 in EV71-associated HFMD are still unclear. HMGB1 expression was detected using RT-qPCR and western blot assays. Loss- and gain-function experiments were performed to evaluate the effects of HMGB1 on EV71-infected cells. The virus titer was examined by TCID50. CCK-8 and flow cytometry assays were applied to detect the cell viability and cell cycle. Oxidative stress was determined by relative commercial kits. HMGB1 level was elevated in the serum of EV71-infected patients with HFMD and EV71-induced RD cells. EV71 infection induced the transfer of HMGB1 from the nucleus into the cytoplasm. HMGB1 knockdown inhibited virus replication,viral protein (VP1) expression and promoted antiviral factor expression. In addition, the inhibition of HMGB1 improved cell viability, protected against S phase arrest, and inhibited EV71-induced cell injury and oxidative stress, whereasHMGB1 overexpression showed the opposite effects. In terms of mechanism, HMGB1 overexpression activated the TLR4/NF-κB/NLRP3 signaling pathway and promoted cell pyroptosis. The inhibition of TLR4 and NF-κB reversed the effects of HMGB1 overexpression on virus replication, oxidative stress, and pyroptosis. In conclusion, HMGB1 knockdown inhibits EV71 replication and attenuates pyroptosis through TLR4/NF-κB/NLRP3 axis.

Network pharmacology-based exploration identified the antiviral efficacy of Quercetin isolated from mulberry leaves against enterovirus 71 via the NF-κB signaling pathway.

Introduction: Mulberry leaf (ML) is known for its antibacterial and anti-inflammatory properties, historically documented in "Shen Nong's Materia Medica". This study aimed to investigate the effects of ML on enterovirus 71 (EV71) using network pharmacology, molecular docking, and in vitro experiments. Methods: We successfully pinpointed shared targets between mulberry leaves (ML) and the EV71 virus by leveraging online databases. Our investigation delved into the interaction among these identified targets, leading to the identification of pivotal components within ML that possess potent anti-EV71 properties. The ability of these components to bind to the targets was verified by molecular docking. Moreover, bioinformatics predictions were used to identify the signaling pathways involved. Finally, the mechanism behind its anti-EV71 action was confirmed through in vitro experiments. Results: Our investigation uncovered 25 active components in ML that targeted 231 specific genes. Of these genes, 29 correlated with the targets of EV71. Quercetin, a major ingredient in ML, was associated with 25 of these genes. According to the molecular docking results, Quercetin has a high binding affinity to the targets of ML and EV71. According to the KEGG pathway analysis, the antiviral effect of Quercetin against EV71 was found to be closely related to the NF-κB signaling pathway. The results of immunofluorescence and Western blotting showed that Quercetin significantly reduced the expression levels of VP1, TNF-α, and IL-1β in EV71-infected human rhabdomyosarcoma cells. The phosphorylation level of NF-κB p65 was reduced, and the activation of NF-κB signaling pathway was suppressed by Quercetin. Furthermore, our results showed that Quercetin downregulated the expression of JNK, ERK, and p38 and their phosphorylation levels due to EV71 infection. Conclusion: With these findings in mind, we can conclude that inhibiting the NF-κB signaling pathway is a critical mechanism through which Quercetin exerts its anti-EV71 effectiveness.

Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

Phosphorylation of ERK-Dependent NF-κB Triggers NLRP3 Inflammasome Mediated by Vimentin in EV71-Infected Glioblastoma Cells.

Enterovirus 71 (EV71) is a dominant pathogenic agent that may cause severe central nervous system (CNS) diseases among infants and young children in the Asia-pacific. The inflammasome is closely implicated in EV71-induced CNS injuries through a series of signaling pathways. However, the activation pathway of NLRP3 inflammasome involved in EV71-mediated CNS injuries remains poorly defined. In the studies, EV71 infection, ERK1/2 phosphorylation, and activation of NLRP3 are abolished in glioblastoma cells with low vimentin expression by CRISPR/Cas9-mediated knockdown. PD098059, an inhibitor of p-ERK, remarkably blocks the vimentin-mediated ERK1/2 phosphorylation in EV71-infected cells. Nuclear translocation of NF-κB p65 is dependent on p-ERK in a time-dependent manner. Moreover, NLRP3 activation and caspase-1 production are limited in EV71-infected cells upon the caffeic acid phenethyl ester (CAPE) administration, an inhibitor of NF-κB, which contributes to the inflammasome regulation. In conclusion, these results suggest that EV71-mediated NLRP3 inflammasome could be activated via the VIM-ERK-NF-κB pathway, and the treatment of the dephosphorylation of ERK and NF-κB inhibitors is beneficial to host defense in EV71-infected CNS.

Structure and antiviral activity of a pectic polysaccharide from the root of Sanguisorba officinalis against enterovirus 71 in vitro/vivo

The increasing prevalence and pandemic risk of viral diseases warrant the development of safe and effective treatments. In this study, we aimed to elucidate the structure and anti-enterovirus 71 (EV71) effects of polysaccharides isolated from the roots of Sanguisorba officinalis (SO), traditionally used for infectious diseases. The purified polysaccharide (S-a3) was a homogenous macromolecule (260.4 kDa) with a concave and porous surface. Linkage and NMR analyses confirmed that S-a3 is a polysaccharide interlinked with homogalacturonan, rhamnogalacturonan-I, 1,4-α-glucan, and arabinogalactan. S-a3 significantly inhibited cell death and viral gene expression in EV71-infected Vero cells, and alleviated EV71-induced body weight loss, death, and paralysis in the hSCARB2-transgenic mouse model. The effective dose of S-a3 was non-toxic to cells and mice. The antiviral mechanism of S-a3 was associated with the disruption of EV71 attachment to host cells. Our findings demonstrate that polysaccharides from SO can be a safe and effective treatment for EV71 infection.

Isolation and Identification of Andrographis paniculata (Chuanxinlian) and Its Biologically Active Constituents Inhibited Enterovirus 71-Induced Cell Apoptosis.

Aim: Andrographis paniculata (Burm. f.) Nees (also known as Chuanxinlian in Chinese) of Acanthaceae family is one of the Chinese herbs reputed to be effective in the treatment of inflammation, infection, cold, and fever. Enterovirus 71 (EV71) is one of the most important enteroviruses that cause hand, foot, and mouth disease (HFMD) accompanied with neurological complication. Methods: To explore an anti-infective Chinese herb medicine, pure compounds isolated or synthesized analogues from A. paniculata (AP) ethyl acetate (EtOAc) extract are used to explore their anti-EV71-induced cytotoxicity. The antiviral activity was determined by cytopathic effect (CPE) reduction, and sub-G1 assays were used for measuring lysis and apoptosis of EV71-infected rhabdomyosarcoma (RD) cells. IFNγ-driven luciferase reporter assay was used to evaluate their potential roles in activation of immune responses. Results: Our data showed that EV71-induced sub-G1 phase of RD cells was dose dependently increased. Highly apoptotic EV71-infected RD cells were reduced by AP extract treatment. Ergosterol peroxide (4) has the most anti-apoptotic effect among these seven compounds. In addition, 3,19-O-acetyl-14-deoxy-11,12-didehydroandrographolide (8) synthesized from acetylation of compound 7 showed significantly better antiviral activity and the lowest sub-G1 phase of 6%–18%. Further investigation of IFNγ-inducer activity of these compounds showed that compounds 3, 6, 10, 11, and 12 had significantly higher IFNγ luciferase activities, suggesting their potential to promote IFNγ expression and thus activate immune responses for antivirus function. Conclusion: Our study demonstrated that bioactive compounds of AP and its derivatives either protecting EV71-infected RD cells from sub-G1 arrest or possessing IFNγ-inducer activity might be feasible for the development of anti-EV71 agents.

Anemoside B4 inhibits enterovirus 71 propagation in mice through upregulating 14-3-3 expression and type I interferon responses.

Enterovirus 71 (EV71) is the major pathogens of human hand, foot, and mouth disease (HFMD). EV71 efficiently escapes innate immunity responses of the host to cause infection. At present, no effective antiviral drugs for EV71 are available. Anemoside B4 (B4) is a natural saponin isolated from the roots of Pulsatilla chinensis (Bunge) Regel. P. chinensis extracts that shows a wide variety of biological activities. In this study, we investigated the antiviral activities of B4 against EV71 both in cell culture and in suckling mice. We showed that B4 (12.5-200 μM) dose dependently increased the viability of EV71-infected RD cells with an IC50 value of 24.95 ± 0.05 μM against EV71. The antiviral activity of B4 was associated with enhanced interferon (IFN)-β response, since knockdown of IFN-β abolished its antiviral activity. We also confirmed that the enhanced IFN response was mediated via activation of retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) pathway, and it was executed by upregulation of 14-3-3 protein, which disrupted the interaction between yes-associated protein (YAP) and interferon regulatory factor 3 (IRF3). By using amino acids in cell culture (SILAC)-based proteomics profiling, we identified the Hippo pathway as the top-ranking functional cluster in B4-treated EV71-infected cells. In vivo experiments were conducted in suckling mice (2-day-old) infected with EV71 and subsequently B4 (200 mg · kg-1 · d-1, i.p.) was administeredfor 16 days. We showed that B4 administration effectively suppressed EV71 replication and improved muscle inflammation and limb activity. Meanwhile, B4 administration regulated the expressions of HFMD biomarkers IL-10 and IFN-γ, attenuating complications of EV71 infection. Collectively, our results suggest that B4 could enhance the antiviral effect of IFN-β by orchestrating Hippo and RLRs pathway, and B4 would be a potential lead compound for developing an anti-EV71 drug.

MiR-103/miR-107 inhibits enterovirus 71 replication and facilitates type I interferon response by regulating SOCS3/STAT3 pathway.

Enterovirus71 (EV71), the major cause of hand, foot, and-mouth disease (HFMD), has increasingly become a public health challenge. Type I interferons (IFNs) can regulate innate and adaptive immune responses to pathogens. MicroRNAs (miRNAs) play regulatory roles in host innate immune responses to viral infections. However, the roles of miR-103 and miR-107 in EV71 infection remain unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of miR-103, miR-107, suppressor of cytokine signaling 3 (SOCS3), VP1, IFN-α, and IFN-β. Virus titers were measured by 50% tissue culture infectious dose (TCID50) assay. Western blot assay was conducted to detect the protein levels of VP1, IFN-α, IFN-β, SOCS3, signal transducer and activator of transcription 3 (STAT3), and phospho-STAT3 (p-STAT3). Immunofluorescence assay was used to detect the protein level of VP1. The concentrations of IFN-α and IFN-β were examined by Enzyme-linked immunosorbent assay (ELISA). The interaction between SOCS3 and miR-103/miR-107 was predicted by starBase and verified by dual-luciferase reporter assay and RNA pull-down assay. MiR-103 and miR-107 were downregulated and SOCS3 was upregulated in serum from patients with EV71 and EV71-infected cells. Overexpression of miR-103 and miR-107 repressed EV71 replication by inhibiting EV71 titers and VP1 expression. Moreover, upregulation of miR-103 and miR-107 enhanced EV71-triggered the production of type I IFNs. In addition, miR-103 and miR-107 directly targeted SOCS3, and SOCS3 upregulation reversed the effects of miR-103 and miR-107 on EV71 replication and type I IFN response. Importantly, miR-103 and miR-107 increased STAT3 phosphorylation by targeting SOCS3 after EV71 infection. MiR-103 and miR-107 suppressed EV71 replication and increased the production of type I IFNs by regulating SOCS3/STAT3 pathway, which might provide a novel strategy for developing effective antiviral therapy.

Viral Interference as a Factor of False-Negative in the Infectious Adenovirus Detection Using Integrated Cell Culture-PCR with a BGM Cell Line

This study investigated the influence of viral interference on the detection of enteric viruses using the integrated cell culture (ICC)-PCR with a BGM cell line. It was possible to detect 102 plaque-forming units (PFU)/flask of enterovirus 71 (EV71) in spite of the presence of 104 PFU/flask of adenovirus 40 (AdV40). Meanwhile, 104 PFU/flask of AdV40 was not detected in the presence of 102 PFU/flask of EV71. This inhibition of AdV40 detection using ICC-PCR was attributable to the growth of EV71, because the addition of a growth inhibitor of EV71 (rupintrivir) neutralized the detection inhibition of AdV40. The growth inhibition of AdV40 under co-infection with EV71 is probably caused by the immune responses of EV71-infected cells. AdV is frequently used as a fecal contamination indicator of environmental water, but this study demonstrated that false-negative detection of infectious AdV using ICC-PCR could be caused by the co-existence of infectious EV in a water sample. The addition of rupintrivir could prevent false-negative detection of AdV using ICC-PCR. This study, therefore, emphasizes the importance of confirming the presence of multiple enteric viruses in a sample derived from environmental water prior to the application of ICC-PCR because the viral interference phenomenon may lead to the false-negative detection of target viruses.

Ginsenoside Rb1 is an immune-stimulatory agent with antiviral activity against enterovirus 71

Ethnopharmacological relevanceAccording to the theory of traditional Chinese medicine, the main pathogenesis of severe hand, foot and mouth disease (HFMD) is that the heat and wet poisons are deeply trapped in the viscera, which causes the deficiency of Qi and Yin in the patient's body. Ginsenoside Rb1 (Rb1) is the most abundant triterpenoid saponin in Panax quinquefolius L., which has the function of Qi-invigorating and Yin-nourishing. Enterovirus 71 (EV71) is one of the causative pathogens of HFMD, especially the form associated with some lethal complications. Therefore, the therapeutic effect of Rb1 on this disease caused by EV71 infection is worth exploring. Aim of the studyWe explored the effective antiviral activities of Rb1 against EV71 in vitro and in vivo and investigated its preliminary antiviral mechanisms. Material and methodsEV71-infected two-day-old suckling mice model was employed to detect the antiviral effects of Rb1 in vivo. To detect the antiviral effects of Rb1 in vitro, cytopathic effect (CPE) reduction assay was performed in EV71-infected Rhabdomyosarcoma (RD) cells. Interferon (IFN)-β interference experiment was employed to detect the antiviral mechanism of Rb1. ResultsIn this paper, we first found that Rb1 exhibited strong antiviral activities in EV71-infected suckling mice when compared to those of ribavirin. Administration of Rb1 reduced the CPE of EV71-infected RD cells in a dose-dependent manner. Moreover, EV71-induced viral protein-1 (VP-1) expression was significantly reduced by Rb1 administration in vitro and in vivo. Furthermore, Rb1 treatment could induce high cellular and humoral immune responses in vivo. Meanwhile, Rb1 contributed to the enhanced Type I IFN responses and IFN-β knockdown reversed the antiviral activity of Rb1 in vitro. ConclusionIn summary, our findings suggest that Rb1 is an immune-stimulatory agent and provide an insight into therapeutic potentials of Rb1 for the treatment of EV71 infection.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Exosomes from EV71-infected oral epithelial cells can transfer miR-30a to promote EV71 infection.

As an extracellular vesicle, exosomes can release from virus-infected cells containing various viral or host cellular elements and could stimulate recipient's cellular response. Enterovirus 71 (EV71), a single-strand positive-sense RNA virus, is known to cause hand, foot, and mouth disease (HFMD) in children and bring about severe clinical diseases. Separated the human oral epithelial cells (OE cells) from normal buccal mucosa through enzyme digestion. Performed a comprehensive miRNA profiling in exosomes from EV71-infected OE cells through deep small RNA-seq. Using the Human Antiviral Response RT Profiler PCR Array profiles to explore the interactions of innate immune signaling networks with exosomal miR-30a. Knocked out the MyD88 gene in macrophages using CRISPR/Cas9-mediated genome editing method. Our study demonstrated that the miR-30a was preferentially enriched in exosomes that released from EV71-infected human oral epithelial cells through small RNA-seq. We found that the transfer of exosomal miR-30a to macrophages could suppress type Ⅰ interferon response through targeting myeloid differentiation factor 88 (MyD88) and subsequently facilitate the viral replication. Exosomes released from EV71-infected OE cells selectively packaged high level of miR-30a that can be functionally transferred to the recipient macrophages resulted in targeting MyD88 and subsequently inhibited type I interferon production in receipt cells, thus promoting the EV71 replication.

EV71 infection induces cell apoptosis through ROS generation and SIRT1 activation.

Several studies have substantiated the correlation between reactive oxygen species (ROS) and Sirtuin 1 (SIRT1). Normally, enterovirus 71 (EV71) is associated with severe clinical manifestations and death. However, the effect of EV71 on the induction of cellular death and the interplay between ROS/SIRT1 in cell death has not been confirmed yet. In the current study, an increase in the number of apoptotic cells was observed as soon as the EV71 infection was initiated in cells and mice. Furthermore, EV71 infection also promoted a rise in the levels of three commonly known proinflammatory cytokines, interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-α. During EV71-induced apoptosis in the different cell lines, ROS generation and SIRT1 downregulation were observed. Further investigations showed that the administration of ROS inhibitor, N-acetyl- l-cysteine (NAC), reduced the level of apoptosis and inflammation, reduced EV71 propagation, and increased SIRT1 expression in EV71-infected cells. In addition, combined administration of NAC and EX527 (SIRT1 inhibitor) restored apoptosis in the EV71-infected cells, which was reduced due to NAC. This data demonstrated that ROS generation is positively associated with EV71-induced apoptosis and inflammation, while this effect could be reversed by SIRT1 inhibition. Collectively, we have shown that EV71 induces apoptosis and inflammation by promoting ROS generation and reducing SIRT1 expression.

EV71 Infection Induces IFNβ Expression in Neural Cells.

Enterovirus 71 (EV71) can invade the central nervous system (CNS) and cause neurological disease. Accumulating evidence indicates that EV71 can directly infect neurons in the CNS. Innate immune responses in the CNS have been known to play an essential role in limiting pathogen infections. Thus, investigating the effects of EV71 infection of neural cells is important for understanding disease pathogenesis. In this study, human neural cells were infected with EV71, and interferonβ (IFNβ) expression was examined. Our results show that IFNβ expression was upregulated in EV71-infected neural cells via pattern recognition receptors (PRRs) sensing of virus RNA. The PRRs Toll-like receptor 3 (TLR3), Toll-like receptor 8 (TLR8), and melanoma differentiation-associated gene-5 (MDA-5), but not retinoic acid-inducible gene-I (RIG-I) and Toll-like receptor 7 (TLR7), were found to be EV71-mediated IFNβ induction. Although viral proteins exhibited the ability to cleave mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF) in neural cells, levels of viral protein expression were low in these cells. Furthermore, neural cells efficiently produced IFNβ transcripts upon EV71 vRNA stimulation. Treating infected cells with anti-IFNβ antibodies resulted in increased virus replication, indicating that IFNβ release may play a role in limiting viral growth. These results indicate that EV71 infection can induce IFNβ expression in neural cells through PRR pathways.

EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production.

As a neurotropic virus, human Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) and may develop severe neurological disorders in infants. Toll-like receptor 7 (TLR7) acts as an innate immune receptor and is also a death receptor in the central nervous system (CNS). However, the mechanisms underlying the regulation of TLR7-mediated brain pathogenesis upon EV71 infection remain largely elusive. Here we reveal a novel mechanism by which EV71 infects astrocytes in the brain and induces neural pathogenesis via TLR7 and interleukin-6 (IL-6) in C57BL/6 mice and in human astroglioma U251 cells. Upon EV71 infection, wild-type (WT) mice displayed more significant body weight loss, higher clinical scores, and lower survival rates as compared with TLR7-/- mice. In the cerebral cortex of EV71-infected mice, neurofilament integrity was disrupted, and inflammatory cell infiltration and neurodegeneration were induced in WT mice, whereas these were largely absent in TLR7-/- mice. Similarly, IL-6 production, Caspase-3 cleavage, and cell apoptosis were significantly higher in EV71-infected WT mice as compared with TLR7-/- mice. Moreover, EV71 preferentially infected and induced IL-6 in astrocytes of mice brain. In U251 cells, EV71-induced IL-6 production and cell apoptosis were suppressed by shRNA-mediated knockdown of TLR7 (shTLR7). Moreover, in the cerebral cortex of EV71-infected mice, the blockade of IL-6 with anti-IL-6 antibody (IL-6-Ab) restored the body weight loss, attenuated clinical scores, improved survival rates, reduced the disruption of neurofilament integrity, decreased cell apoptotic induction, and lowered levels of Caspase-3 cleavage. Similarly, in EV71-infected U251 cells, IL-6-Ab blocked EV71-induced IL-6 production and cell apoptosis in response to viral infection. Collectively, it’s exhibited TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Taken together, we propose that EV71 infects astrocytes of the cerebral cortex in mice and human and triggers TLR7 signaling and IL-6 release, subsequently inducing neural pathogenesis in the brain.

Salvianolic Acid B Inhibits Hand-Foot-Mouth Disease Enterovirus 71 Replication through Enhancement of AKT Signaling Pathway.

Hand, foot, and mouth disease (HFMD) is caused by enterovirus 71 (EV71) in infants and children under six years of age. HFMD is characterized by fever, mouth ulcers, and vesicular rashes on the palms and feet. EV71 also causes severe neurological manifestations, such as brainstem encephalitis and aseptic meningitis. Recently, frequent outbreaks of EV71 have occurred in the Asia-Pacific region, but currently, no effective antiviral drugs have been developed to treat the disease. In this study, we investigated the antiviral effect of salvianolic acid B (SalB) on EV71. SalB is a major component of the Salvia miltiorrhiza root and has been shown to be an effective treatment for subarachnoid hemorrhages and myocardial infarctions. HeLa cells were cultured in 12-well plates and treated with SalB (100 or 10 µg/ml) and 106 PFU/ml of EV71. SalB treatment (100 µg/ml) significantly decreased the cleavage of the eukaryotic eIF4G1 protein and reduced the expression of the EV71 capsid protein VP1. In addition, SalB treatment showed a dramatic decrease in viral infection, measured by immunofluorescence staining. The Akt signaling pathway, a key component of cell survival and proliferation, was significantly increased in EV71-infected HeLa cells treated with 100 µg/ml SalB. RT-PCR results showed that the mRNA for anti-apoptotic protein Bcl-2 and the cell cycle regulator Cyclin-D1 were significantly increased by SalB treatment. These results indicate that SalB activates Akt/PKB signaling and inhibits apoptosis in infected HeLa cells. Taken together, these results suggest that SalB could be used to develop a new therapeutic drug for EV71-induced HFMD.

Functionalized selenium nanoparticles enhance the anti-EV71 activity of oseltamivir in human astrocytoma cell model

Enterovirus 71 (EV71) which commonly caused the hand-foot-mouth disease (HFMD) has become one of public health challenges worldwide. However, no effective vaccines or drugs for this disease has been developed. Thus, there is an urgent need to find a new strategy for treating the EV71 infection. Oseltamivir (OT) is an effective antiviral agent, but continuous use of oseltamivir leads to a diminished therapeutic effect in the clinic. In order to improve the antiviral activity of oseltamivir, oseltamivir was loaded onto surfaces of selenium nanoparticles (SeNPs) to fabricate a functionalized antiviral nanoparticles SeNPs@OT. The size of SeNPs@OT was tested by TEM and dynamic light scattering. The chemical structure and elemental composition of SeNPs@OT were analyzed by FT-IR and EDX, respectively. SeNPs@OT exhibited good stability and effective drug release in serum and PBS. SeNPs@OT efficiently entered into human astrocyte U251 cells (host cells) via clathrin-associated endocytosis and inhibited EV71 proliferation, which could protect EV71-infected U251 cells from apoptosis through mitochondrial pathway. Furthermore, SeNPs@OT inhibited EV71 activity probably by reducing the generation of reactive oxygen species in EV71-infected U251 cells. Interestingly, SeNPs obviously enhanced antiviral activity of oseltamivir in the anti-EV71 cell model. Taken together, SeNPs@OT is a promising antiviral drug candidate for EV71 infection.

Hederacolchiside C inhibits Enterovirus 71 propagation through activating innate immunity

Enterovirus 71 (EV71), a newly emerging life-threatening pathogen induces hand-foot-mouth disease (HFMD), no effective vaccines or specific anti-viral treatments are currently available. In this study, the activity of hederacolchiside C (HSC) against EV71 was investigated, and the antiviral mechanism was explored. HSC displayed apparent antiviral activity in EV71-infected cells probably through activating the host innate immunity. Comparing with EV71-infected group at 24 hpi, the group pretreated with HSC dramatically increased the expression of MAVS, p-IRF3, IRF3 and IFN-β, the innate immune effectors related to innate immunity. In addition, HSC displayed stronger antiviral activity in EV71-infected suckling mice in comparison with Ribavirin, a broad-spectrum antiviral drug. The results suggest that HSC could have potential as a pharmaceutical drug for HFMD.