Objective To investigate the role and mechanism of RNA-Specific adenosine deaminase 3 (Adar3) in regulating macrophage polarization during Coxsackievirus B3(CVB3)-induced viral myocarditis (VM). Methods Bone marrow-derived macrophages (BMDM) from mice were cultured in vitro and induced into M1/M2 macrophages using interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) or interleukin 4 (IL-4), respectively. The mRNA expression levels of Adar1, Adar2, and Adar3 in each group of cells were assessed by real-time quantitative PCR (qRT-PCR). Specific siRNAs targeting the Adar3 gene were designed, synthesized, and transiently transfected into M2 macrophages. The mRNA levels of M2 polarization-related marker genes-including arginase 1 (Arg1), chitinase 3-like molecule 3 (YM1/Chi3l3), and resistin-like molecule alpha (RELMα/FIZZ1)-were detected by qRT-PCR. RNA sequencing was performed to analyze the signaling pathways affected by Adar3. The expression levels of Wnt/β-catenin signaling pathway were further validated using qRT-PCR and Western blot. The adeno-associated virus overexpressing Adar3 was designed, synthesized, and injected into mice via tail vein. Three weeks later, a myocarditis mouse model was established. After an additional week, the phenotype and function of cardiac macrophages, as well as multiple indicators of VM (including echocardiography, body weight, histopathology and serology) were examined. Additionally, the protein levels of the Wnt/β-catenin signaling pathway were assessed. Results Compared to M0-type macrophages, the expression level of Adar3 was significantly increased in M2-type macrophages. After transfection of Adar3 siRNA, the mRNA levels of Arg1, YM1 and FIZZ1 in M2 macrophages were downregulated. RNA sequencing revealed 149 upregulated genes and 349 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and subsequent validation experiments indicated that Adar3 modulated the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that Adar3 overexpression alleviated the cardiac dysfunction of VM mice. The proportion of M1 macrophages in the heart decreased, while the proportion of M2 macrophages increased. At the same time, the Adar3 overexpression activated the Wnt/β-catenin signaling pathway. Conclusion Adar3 promotes macrophage polarization toward the M2 phenotype by activating the Wnt/β-catenin signaling pathway, thereby alleviating VM.
Animals ; Adenosine Deaminase/metabolism* ; Macrophages/immunology* ; Wnt Signaling Pathway/genetics* ; Myocarditis/immunology* ; Mice ; Coxsackievirus Infections/metabolism* ; Male ; Mice, Inbred BALB C ; Enterovirus B, Human/physiology* ; beta Catenin/genetics*

OBJECTIVES: To observe the therapeutic effect of spermine in neonatal mouse models of severe hand, foot and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection and explore its therapeutic mechanism in light of regulation of macrophage GBP5/NLRP3 inflammasome pathway. METHODS: Neonatal BALB/c mice (3-5 days old) were divided into control group, EV71 infection group and Spermine treatment group. The mice in the latter two groups received an intraperitoneal injection of 50 μL EV71 suspension (1×10⁶ TCID50 of EV71), followed 3 days later by intraperitoneal injection of 50 μL PBS or 100 μmol/L spermine. GBP5, NLRP3, CXCL10, and TNFSF10 expressions in heart, liver, lung and kidney tissues of the mice were detected using Western blotting and qPCR, and tissue pathologies and macrophage infiltration were assessed with HE staining and immunohistochemistry. In cultured THP-1 and RAW264.7 cells, the effects of EV71 infection, GBP5 siRNA transfection and treatment with spermine or eflornithine on GBP5, NLRP3, CXCL10, and TNFSF10 mRNA expressions were investigated using qPCR. RESULTS: In the neonatal mice, EV71 infection resulted in multiple organ damage, macrophage infiltration and activation of the GBP5/NLRP3 pathway, and spermine treatment significantly improved tissue injuries, reduced macrophage infiltration, and down-regulated the expressions of GBP5, NLRP3 and the inflammatory factors in the infected mice. In THP-1 and RAW264.7 cells, EV71 infection caused significant upregulation of GBP5, NLRP3, CXCL10, and TNFSF10 expressions, which were obviously lowered by spermine treatment. In THP-1 cells, treatment with eflornithine significantly suppressed the reduction of GBP5, NLRP3, CXCL10, and TNFSF10 expressions induced by GBP5 siRNA transfection. CONCLUSIONS Spermine suppressed EV71 infection-induced inflammatory responses by inhibiting GBP5-mediated NLRP3 inflammasome activation, suggesting a new strategy for treatment of severe HFMD.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Mice ; Macrophages/metabolism* ; Enterovirus A, Human ; Mice, Inbred BALB C ; Inflammasomes/metabolism* ; Spermine/therapeutic use* ; Animals, Newborn ; Humans ; Enterovirus Infections ; Hand, Foot and Mouth Disease/drug therapy* ; RAW 264.7 Cells ; Chemokine CXCL10/metabolism*

OBJECTIVE: Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3 (CVB3) infection induced autophagy through endoplasmic reticulum (ER) stress. METHODS: In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction (RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase (PERK) inhibitor, inositol-requiring protein-1 (IRE1) inhibitor, or activating transcription factor-6 (ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3 (LC3). RESULTS: CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein (GFP)-LC3 punctuation and induced the conversion from LC3-I to phosphatidylethanolamine-conjugated LC3-1 (LC3-II). CVB3 infection still decreased the expression of mammalian target of rapamycin (mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-II to LC3-I in CVB3-infected HeLa cells. CONCLUSION CVB3 infection induced autophagy through ER stress in HeLa cells, and PERK, IRE1, and ATF6a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.
Activating Transcription Factor 6 ; metabolism ; Autophagy ; Coxsackievirus Infections ; metabolism ; Endoplasmic Reticulum Stress ; Endoribonucleases ; metabolism ; Enterovirus B, Human ; HeLa Cells ; Humans ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; eIF-2 Kinase ; metabolism

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.
Animals ; Cytopathogenic Effect, Viral ; Enterovirus A, Human ; genetics ; pathogenicity ; Enterovirus Infections ; pathology ; virology ; Gene Expression Regulation, Viral ; Mice ; Mice, Inbred ICR ; Mutation ; Viral Load ; Viral Proteins ; genetics ; metabolism ; Virulence ; Virus Replication

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

Enterovirus 71 frequently involves the central nervous system and may present with a variety of neurologic manifestations. Here, we aimed to describe the clinical features, magnetic resonance imaging (MRI) findings, and cerebrospinal fluid (CSF) profiles of patients presenting with neurologic complications of enterovirus 71 infection. We retrospectively reviewed the records of 31 pediatric patients hospitalized with acute neurologic manifestations accompanied by confirmed enterovirus 71 infection at Ulsan University Hospital between 2010 and 2014. The patients' mean age was 2.9 ± 5.5 years (range, 18 days to 12 years), and 80.6% of patients were less than 4 years old. Based on their clinical features, the patients were classified into 4 clinical groups: brainstem encephalitis (n = 21), meningitis (n = 7), encephalitis (n = 2), and acute flaccid paralysis (n = 1). The common neurologic symptoms included myoclonus (58.1%), lethargy (54.8%), irritability (54.8%), vomiting (48.4%), ataxia (38.7%), and tremor (35.5%). Twenty-five patients underwent an MRI scan; of these, 14 (56.0%) revealed the characteristic increased T2 signal intensity in the posterior region of the brainstem and bilateral cerebellar dentate nuclei. Twenty-six of 30 patients (86.7%) showed CSF pleocytosis. Thirty patients (96.8%) recovered completely without any neurologic deficits; one patient (3.2%) died due to pulmonary hemorrhage and shock. In the present study, brainstem encephalitis was the most common neurologic manifestation of enterovirus 71 infection. The characteristic clinical symptoms such as myoclonus, ataxia, and tremor in conjunction with CSF pleocytosis and brainstem lesions on MR images are pathognomonic for diagnosis of neurologic involvement by enterovirus 71 infection.
Acute Disease ; Brain/diagnostic imaging ; Central Nervous System Diseases/etiology/*pathology ; Child ; Child, Preschool ; Encephalitis/pathology ; Enterovirus A, Human/genetics/*isolation & purification ; Enterovirus Infections/drug therapy/*pathology/virology ; Feces/virology ; Female ; Humans ; Immunoglobulins/administration & dosage ; Infant ; Injections, Intravenous ; Leukocytes/cytology ; Leukocytosis/cerebrospinal fluid/pathology ; Magnetic Resonance Imaging ; Male ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Retrospective Studies ; Seasons

OBJECTIVETo investigate the expression of Toll-like receptor (TLR) mRNA in enterovirus 71(EV-A71) infected human Jurkat T cells and clarify the role of TLRs in the pathogenesis of EV-A71 infection-induced inflammation.

METHODSEV-A71 strains were isolated from feces of children patients with hand, foot and mouth disease in 2014 by Shenzhen Center for Disease Control and Prevention. Human Jurkat T cells were infected with 200 μl EV-A71 at 10(3) cell culture infective dose 50%(CCID50)/ml. The expression of TLR1-TLR10 mRNA in human Jurkat T cells was assessed at different exposure time by RT-PCR. Levels of TLR7 mRNA expression were detected by real-time PCR, and levels of myeloid differentiation factor 88 (MyD88) by western blot. The cytokine secretion of interleukin (IL)-6, IL-8 and Tumor Necrosis Factor α (TNF-α) was analyzed by ELISA assay.

RESULTSThe relative expression level of TLR7 mRNA in human Jurkat T cells were 1.26 ± 0.15, 1.75 ± 0.20, 2.26 ± 0.23 and 3.74 ± 0.62 in 6, 12, 24 and 48 h after EV-A71 infection, which the differences were significant with mock-infected group(t values were -2.96, -6.38, -9.57, -7.71; P<0.05). Western blot showed that the protein expression levels of MyD88 had increased 1.34 times and 2.17 times in 24 h and 48 h after EV-A71 infection compared with mock-infected group. After infected for 24 h and 48 h, the levels of IL-6 were (302.86 ± 38.11), (179.70 ± 14.50) pg/ml, which were significantly higher than mock-infected group (176.42 ± 9.60), (179.70 ± 14.50) pg/ml (t values were -5.57, -18.54, P<0.05). The levels of TNF-α in EV-A71 infected group (100.81 ± 9.81) pg/ml was higher than that in mock-infected group (56.19 ± 6.94) pg/ml, and the difference was significant (t=-6.43, P=0.003).

CONCLUSIONTLR7 is the main pattern recognition receptor responsible for EV-A71 recognition in immune cells, which then leads to the activation of TLR7 downstream signaling and the production of proinflammatory cytokines.

Blotting, Western ; Cell Line ; Enterovirus A, Human ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Myeloid Differentiation Factor 88 ; metabolism ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; virology ; Toll-Like Receptor 7 ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the dynamic expression and role of vitamin D receptor (VDR) in the myocardium of mice with viral myocarditis (VMC).

METHODSOne hundred and twenty 4-week-old male BALB/c mice were selected and assigned into control (n=40) and experimental groups (n=80). The mice in the experimental group were injected intraperitoneally with Coxsackievirus B3 to establish the model of VMC, while the mice in the control group were injected intraperitoneally with an equal volume of DMEM solution. Fifteen mice in the experimental group and ten mice in the control group were sacrificed at 3, 7, 14, or 28 days after injection, and the myocardial specimens were obtained. The dynamic expression of VDR in the myocardium was determined by the immunohistochemical technique. The pathological changes in the myocardium were examined using hematoxylin and eosin staining.

RESULTSIn the experimental group, the mice had significantly increased expression of VDR after virus injection (P<0.01); the expression of VDR reached the peak at 7 days after injection, and then declined gradually; the expression of VDR remained high at 28 days after injection. At 3, 7, 14, and 28 days after injection, the expression of VDR in the experimental group was significantly higher than that in the control group (P<0.01). Moreover, in the experimental group, the changes in the pathological score of the myocardium were in accordance with the changes in the expression of VDR; the expression level of VDR in the myocardium was positively correlated with the pathological changes in the myocardium in the experimental group (P<0.01).

CONCLUSIONSVDR may be involved in the inflammatory-immune process in the pathogenesis of VMC.

Animals ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; pathology ; Myocardium ; chemistry ; pathology ; Receptors, Calcitriol ; analysis ; physiology

Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Animals ; Carrier Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; Heterogeneous-Nuclear Ribonucleoprotein K ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication