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1.
Cell Physiol Biochem ; 53(1): 121-140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230428

RESUMO

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons.


Assuntos
Infecções por Coxsackievirus/patologia , Enterovirus Humano B/fisiologia , Animais , Proteínas do Capsídeo/metabolismo , Infecções por Coxsackievirus/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Enterovirus Humano B/patogenicidade , Humanos , Canal de Potássio KCNQ1/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Proteínas não Estruturais Virais/metabolismo
2.
Medicine (Baltimore) ; 98(19): e15629, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31083261

RESUMO

OBJECTIVE: To determine the effects and mechanism of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, CC1)-mediated regulation of the Coxsackie and Adenovirus Receptor (CAR) after Coxsackievirus B3 (CVB3) infection. METHODS: A mouse CC1 overexpression recombinant virus was constructed, followed by insertion of a pLVX-CEACAM 1-zsgreen-puro (rLV-CEACAM 1) plasmid into the recombinant retrovirus. Cardiac myocytes were assigned into different groups according to various treatments. The apoptosis rate and cell activity in each group were observed. Further, CAR expression and SYK, IL-1ß, and p-SYK levels were measured. RESULTS: The recombinant retrovirus titer was measured as 1.5 × 10 TUs/ml. The apoptosis rate of cardiac myocytes in the CC1 overexpression plus CVB3 group was significantly elevated, and the relative expression of the CAR gene was the highest in the CC1 overexpression plus CVB3 group. TNF-α and IL-1ß levels increased due to CC1 overexpression and further increased after CVB3 infection. CAR protein expression also changed along with the levels of CC1, SYK, and TNF-α after infection. CONCLUSION: CC1 may promote CAR expression after CVB3 infection and regulate CAR protein expression by activating the CC1-SYK-TNF-α signaling axis during the infection process.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Infecções por Coxsackievirus/metabolismo , Cardiopatias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Apoptose/fisiologia , Antígeno Carcinoembrionário/genética , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Cardiopatias/etiologia , Cardiopatias/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células Musculares/metabolismo , Células Musculares/patologia , Organismos Livres de Patógenos Específicos , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
3.
Basic Res Cardiol ; 114(2): 11, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30673858

RESUMO

Coxsackieviruses of group B (CVB) are well-known causes of acute and chronic myocarditis. Chronic myocarditis can evolve into dilated cardiomyopathy (DCM) characterized by fibrosis and cardiac remodeling. Interleukin-1ß (IL-1ß) plays a decisive role in the induction of the inflammatory response as a consequence of viral replication. In this study, we analyzed the effects of IL-1ß neutralization on the transition of acute to chronic myocarditis in a mouse model of CVB3 myocarditis. Mice were treated with an anti-murine IL-1ß antibody as a surrogate for Canakinumab at different time points post CVB3 infection. Treatment was performed in the early phase (day 1-14 pi, day 3-14 pi) or at a later stage of myocarditis (day 14-28 pi). Subsequently, the hearts were examined histologically, immunohistochemically and by molecular biology. A significant reduction of viral replication, cardiac damage and inflammation was found after administration of the antibody in the early phase and in the later phase of infection. Furthermore, less collagen I deposition and a considerable reduction of fibrosis were found in antibody-treated mice. Using microarray analysis, a significant upregulation of various extracellular matrix and fibrosis-associated molecules was found in CVB3-infected mice, including TGF-ß, TIMP-1 and MMP12, as well as diverse matricellular proteins, whereas, these molecules were significantly downregulated in all IL-1ß antibody-treated infected mice. Neutralization of IL-1ß at different stages of enteroviral infection prevents the development of chronic viral myocarditis by reducing inflammation, interstitial fibrosis and adverse cardiac remodeling. These findings are relevant for the treatment of patients with acute and chronic myocarditis.


Assuntos
Interleucina-1beta/antagonistas & inibidores , Miocardite/patologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Doença Crônica , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B , Camundongos , Miocardite/metabolismo , Miocardite/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
4.
Int J Mol Sci ; 20(2)2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669342

RESUMO

Adenosine deaminases acting on RNA (ADAR) are enzymes that regulate RNA metabolism through post-transcriptional mechanisms. ADAR1 is involved in a variety of pathological conditions including inflammation, cancer, and the host defense against viral infections. However, the role of ADAR1p150 in vascular disease remains unclear. In this study, we examined the expression of ADAR1p150 and its role in viral myocarditis (VMC) in a mouse model. VMC mouse cardiomyocytes showed significantly higher expression of ADAR1p150 compared to the control samples. Coimmunoprecipitation verified that ADAR1p150 forms a complex with Dicer in VMC. miRNA-222, which is involved in many cardiac diseases, is highly expressed in cardiomyocytes in VMC. In addition, the expression of miRNA-222 was promoted by ADAR1p150/Dicer. Among the target genes of miRNA-222, the expression of phosphatase-and-tensin (PTEN) protein was significantly reduced in VMC. By using a bioinformatics tool, we found a potential binding site of miRNA-222 on the PTEN gene's 3'-UTR, suggesting that miRNA-222 might play a regulatory role. In cultured cells, miR-222 suppressed PTEN expression. Our findings suggest that ADAR1p150 plays a key role in complexing with Dicer and promoting the expression of miRNA-222, the latter of which suppresses the expression of the target gene PTEN during VMC. Our work reveals a previously unknown role of ADAR1p150 in gene expression in VMC.


Assuntos
Adenosina Desaminase/metabolismo , Infecções por Coxsackievirus/complicações , Enterovirus Humano B , MicroRNAs/genética , Miocardite/etiologia , Miocardite/metabolismo , PTEN Fosfo-Hidrolase/genética , Ribonuclease III/metabolismo , Animais , Sobrevivência Celular/genética , Células Cultivadas , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Enterovirus Humano B/fisiologia , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Imuno-Histoquímica , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , Ligação Proteica , Interferência de RNA
5.
Biochim Biophys Acta Mol Basis Dis ; 1864(8): 2579-2589, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29730342

RESUMO

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.


Assuntos
Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Musculares/metabolismo , Miocardite/metabolismo , NF-kappa B/metabolismo , Doença Aguda , Animais , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Miocardite/patologia , Miocardite/virologia , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo
6.
Zool Res ; 39(1): 52-57, 2018 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-29511145

RESUMO

Globally, coxsackievirus B4 (CV-B4) has been continuously isolated and evidence suggests an association with the development of pancreatitis and type I diabetes. In addition, CV-B4 is also associated with myocarditis and severe central nervous system (CNS) complications, which remain poorly studied and understood. In the present study, we established an Institute for Cancer Research (ICR) mouse model of CV-B4 infection and examined whether CV-B4 infection resulted in a predisposition to myocarditis and CNS infection. We found high survival in both the treatment and control group, with no significant differences in clinical outcomes observed. However, pathological lesions were evident in both brain and heart tissue of the CV-B4-infected mice. In addition, high viral loads were found in the neural and cardiac tissues as early as 2 days post infection. Expressions of IFN-γ and IL-6 in sera were significantly higher in CV-B4-infected mice compared to uninfected negative controls, suggesting the involvement of these cytokines in the development of histopathological lesions. Our murine model successfully reproduced the acute myocarditis and cerebral cortical neuron edema induced by CV-B4, and may be useful for the evaluation of vaccine candidates and potential antivirals against CV-B4 infection.


Assuntos
Edema Encefálico/virologia , Infecções por Coxsackievirus/complicações , Modelos Animais de Doenças , Enterovirus Humano B , Miocardite/virologia , Animais , Edema Encefálico/etiologia , Edema Encefálico/patologia , Infecções por Coxsackievirus/patologia , Citocinas/sangue , Camundongos , Camundongos Endogâmicos ICR , Miocardite/etiologia , Miocardite/patologia , Neurônios/patologia , Carga Viral
7.
PLoS Pathog ; 14(1): e1006872, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29360865

RESUMO

Cathepsin B (CatB) is a cysteine proteolytic enzyme widely expressed in various cells and mainly located in the lysosomes. It contributes to the pathogenesis and development of many diseases. However, the role of CatB in viral myocarditis (VMC) has never been elucidated. Here we generated the VMC model by intraperitoneal injection of coxsackievirus B3 (CVB3) into mice. At day 7 and day 28, we found CatB was significantly activated in hearts from VMC mice. Compared with the wild-type mice receiving equal amount of CVB3, genetic ablation of CatB (Ctsb-/-) significantly improved survival, reduced inflammatory cell infiltration, decreased serum level of cardiac troponin I, and ameliorated cardiac dysfunction, without altering virus titers in hearts. Conversely, genetic deletion of cystatin C (Cstc-/-), which markedly enhanced CatB levels in hearts, distinctly increased the severity of VMC. Furthermore, compared with the control, we found the inflammasome was activated in the hearts of wild-type mice with VMC, which was attenuated in the hearts of Ctsb-/- mice but was further enhanced in Cstc-/- mice. Consistently, the inflammasome-initiated pyroptosis was reduced in Ctsb-/- mice hearts and further increased in Cstc-/- mice. These results suggest that CatB aggravates CVB3-induced VMC probably through activating the inflammasome and promoting pyroptosis. This finding might provide a novel strategy for VMC treatment.


Assuntos
Catepsina B/fisiologia , Infecções por Coxsackievirus/complicações , Enterovirus Humano B/fisiologia , Inflamassomos/metabolismo , Miocardite/virologia , Piroptose/fisiologia , Animais , Caspase 1/metabolismo , Catepsina B/genética , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/imunologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia
8.
Mol Cell Biochem ; 442(1-2): 11-18, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28887702

RESUMO

In this study, we investigated the roles of RIP1/RIP3 mediated cardiomyocyte necroptosis in CVB3-induced acute myocarditis. Serum concentrations of creatinine kinase (CK), CK-MB, and cardiac troponin I were detected using a Hitachi Automatic Biochemical Analyzer in a mouse model of acute VMC. Histological changes in cardiac tissue were observed by light microscope and expression levels of RIP1/RIP3 in the cardiac tissue were detected via Western blot and immunohistochemistry. The data showed that RIP1/RIP3 was highly expressed in cardiomyocytes in the acute VMC mouse model and that the necroptosis pathway specific blocker, Nec-1, dramatically reduced the myocardial damage by downregulating the expression of RIP1/RIP3. These findings provide evidence that necroptosis plays a significant role in cardiomyocyte death and it is a major pathway for cell death in acute VMC. Blocking the necroptosis pathway may serve as a new therapeutic option for the treatment of acute viral myocarditis.


Assuntos
Miocardite/metabolismo , Miócitos Cardíacos/metabolismo , Doença Aguda , Animais , Morte Celular , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/patologia , Miocardite/virologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
9.
PLoS Pathog ; 13(12): e1006744, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29220410

RESUMO

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.


Assuntos
Infecções por Coxsackievirus/virologia , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Miocardite/virologia , Miócitos Cardíacos/virologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/fisiologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos A , Mutação , Miocardite/imunologia , Miocardite/metabolismo , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Replicação Viral
10.
Hawaii J Med Public Health ; 76(11 Suppl 2): 3-6, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29164007

RESUMO

Adult-onset Still's Disease is a rare, idiopathic, inflammatory disorder characterized by arthralgia, evanescent, salmon-colored rash, and daily fevers as well as lymphadenopathy, pharyngitis, splenomegaly, myalgias, and serositis. The inciting etiology of this syndrome is unknown, though it has been hypothesized that infection triggers an autoimmune response. The Yamaguchi Criteria, the most sensitive and widely used diagnostic criteria, requires both a minimum set of criteria to be met as well other potential etiologies to be excluded. By definition, evidence of concomitant infection, malignancy, vasculitis, or connective tissue disease precludes the diagnosis of Adult-onset Still's Disease from being made. We present a very rare case of a patient who met all diagnostic criteria for Adult-onset Still's Disease, had a protracted course refractory to numerous immunosuppressant treatments, and also had evidence of coxsackie B infection with fourfold rise in viral titers on two occasions (both associated with disease flare). Although coxsackie B virus has been linked to Adult-onset Still's Disease at disease presentation, this case is unique in its protracted course and serological evidence of infection temporally related to disease flare. While accepted diagnostic criteria call for this disease to be a diagnosis of exclusion, our case supports the fact that ongoing infection may in fact be an important antigenic driver in persistent and refractory Adult-onset Still's Disease.


Assuntos
Doença de Still de Início Tardio/diagnóstico , Adulto , Infecções por Coxsackievirus/diagnóstico , Infecções por Coxsackievirus/patologia , Diagnóstico Diferencial , Enterovirus Humano B , Humanos , Masculino , Doença de Still de Início Tardio/patologia , Sinovite/diagnóstico por imagem , Sinovite/patologia
11.
PLoS One ; 12(8): e0182643, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28800592

RESUMO

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.


Assuntos
Quimiocina CX3CL1/imunologia , Infecções por Coxsackievirus/genética , Enterovirus Humano B/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Miocardite/genética , Receptores de Quimiocinas/imunologia , Animais , Apoptose , Receptor 1 de Quimiocina CX3C , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Quimiocina CX3CL1/genética , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Enterovirus Humano B/crescimento & desenvolvimento , Regulação da Expressão Gênica , Testes de Função Cardíaca , Humanos , Interleucinas/genética , Interleucinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/imunologia , Miocardite/patologia , Miocardite/virologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética
13.
Nat Microbiol ; 2: 17088, 2017 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-28581455

RESUMO

RNA viruses pose serious threats to human health. Their success relies on their capacity to generate genetic variability and, consequently, on their adaptive potential. We describe a strategy to attenuate RNA viruses by altering their evolutionary potential. We rationally altered the genomes of Coxsackie B3 and influenza A viruses to redirect their evolutionary trajectories towards detrimental regions in sequence space. Specifically, viral genomes were engineered to harbour more serine and leucine codons with nonsense mutation targets: codons that could generate Stop mutations after a single nucleotide substitution. Indeed, these viruses generated more Stop mutations both in vitro and in vivo, accompanied by significant losses in viral fitness. In vivo, the viruses were attenuated, generated high levels of neutralizing antibodies and protected against lethal challenge. Our study demonstrates that cornering viruses in 'risky' areas of sequence space may be implemented as a broad-spectrum vaccine strategy against RNA viruses.


Assuntos
Adaptação Biológica , Códon sem Sentido , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidade , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Mutação Puntual , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Códon , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Virulência
14.
Antiviral Res ; 144: 247-255, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28625478

RESUMO

Epidemiological data indicate that coxsackievirus A10 (CVA10) has become one of the main causative agents of hand, foot and mouth disease (HFMD) and in recent years has often been found to co-circulate with other enteroviruses, which poses a challenge for the prevention and control of HFMD. Although most CVA10-associated HFMD cases present mild symptoms, severe manifestations and even death can also occur. However, the study of the pathogenesis and the development of drugs and vaccines for CVA10 infection are still far from complete. In this study, we established a neonatal mouse model for anti-viral evaluation and characterized the pathology of CVA10 infection. To develop the mouse model, both inbred and outbred mouse strains were used to compare their sensitivity to CVA10 infection; then, one-day-old BALB/c mice were selected and inoculated intraperitoneally with a CVA10 clinical strain, CVA10-FJ-01. Clinical symptoms, such as wasting, hind-limb paralysis and even death were observed in the CVA10-infected mice. Moreover, pathological examination and immunohistochemistry staining showed that severe myonecrosis with inflammatory infiltration was observed in CVA10-infected mice, indicating that CVA10 exhibited strong tropism to muscle tissue. Using real-time PCR, we also found that the viral load in the blood and muscle was higher than that in other organs/tissues at different time points post-infection, suggesting that CVA10 had a strong tropism to mice muscle and that viremic spread may also contribute to the death of the CVA10-infected mice. Additionally, to evaluate the neonatal mouse model of CVA10 infection, female mice were immunized with formalin-inactivated CVA10 and then allowed to mate after the third immunization. The results showed that maternal antibodies could protect mice against CVA10 infection. In summary, the results demonstrated that the neonatal mice model was a useful tool for evaluating the protective effects of CVA10 vaccines and anti-viral reagents.


Assuntos
Antivirais/administração & dosagem , Infecções por Coxsackievirus/tratamento farmacológico , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Enterovirus/patogenicidade , Animais , Animais Recém-Nascidos , Sangue/virologia , Infecções por Coxsackievirus/virologia , Camundongos Endogâmicos BALB C , Miosite/patologia , Miosite/virologia , Necrose/patologia , Carga Viral , Tropismo Viral
15.
Rev. esp. patol ; 50(1): 8-14, ene.-mar. 2017. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-159058

RESUMO

A pesar de que las infecciones por virus de la familia Herpesviridae son muy frecuentes, tan solo 8 subtipos afectan al hombre (virus herpes simplex tipos 1 y 2, virus varicela-zoster, virus Epstein-Barr, citomegalovirus y virus herpes humano tipos 6, 7 y 8). Entre las infecciones causadas por enterovirus destacan el Poliovirus, el Coxsackievirus y el Echovirus. La clínica de estas infecciones puede variar desde un cuadro leve a una afectación sistémica grave, siendo importante el diagnóstico precoz. En la actualidad, la técnica de arrays de baja densidad es capaz de detectar diferentes virus en un mismo análisis a partir de ADN extraído de muestras biológicas. En nuestro estudio empleamos el kit CLART® ENTHERPEX, con el que analizamos 70 muestras de tejido fijado en formol y parafinado, investigando la presencia de virus (VHS-1, VHS-2, VVZ, CMV, VEB, VHH-6, VHH-7 y VHH-8, Poliovirus, Echovirus y Coxsackievirus). De las 70 muestras analizadas, en 29 (41,43%) se detectó infección vírica; solo 4 de las muestras positivas presentaban lesión citopática (correlación del 100% entre el análisis histológico y el test). Las muestras de EICH mostraron un 47,6% de positividad, en EII se detectaron un 68,75% de positivos, en colitis no EII con úlceras se detectó un 50% de positividad, y un 50% de positividad en las lesiones isquémicas. La técnica empleada tiene una alta sensibilidad, por lo que sería útil realizar la determinación como ayuda al diagnóstico basado en la histología, ya que en ocasiones la presencia de infección vírica puede modificar el manejo terapéutico (AU)


Despite the frequency of infections with herpesviridae family, only eight subtypes affect humans (Herpex Simplex Virus types 1 and 2, Varicella Zoster Virus, Epstein-Barr Virus, Citomegalovirus and Human Herpes Virus types 6, 7 and 8). Amongst enteroviruses infections, the most important are Poliovirus, Coxackievirus and Echovirus. Symptoms can vary from mild to severe and early diagnosis is of upmost importance. Nowadays, low-density arrays can detect different types of viruses in a single assay using DNA extracted from biological samples. We analyzed 70 samples of formalin-fixed and paraffin-embedded tissue, searching for viruses (HSV-1, HSV-2, VZV, CMV, EBV, HHV-6, HHV-7 y HHV-8, Poliovirus, Echovirus and Coxsackievirus) using the kit CLART® ENTHERPEX. Out of the total of 70 samples, 29 were positive for viral infection (41.43%), and only 4 of them showed cytopathic effect (100% correlation between histology and the test). 47.6% of GVHD samples were positive for virus; 68.75% of IBD analyzed showed positivity for viral infection; in colitis with ulcers (neither GVHD nor IBD), the test was positive in 50% of the samples and was also positive in 50% of ischemic lesions. The high sensitivity of the technique makes it a useful tool for the pathologist in addition to conventional histology-based diagnosis, as a viral infection may affect treatment (AU)


Assuntos
Feminino , Gravidez , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Humanos , Infecções por Herpesviridae/diagnóstico , Enterovirus/isolamento & purificação , Diagnóstico Precoce , Poliovirus , Poliovirus/isolamento & purificação , Infecções por Coxsackievirus/patologia , Infecções por Echovirus/patologia , Análise em Microsséries/métodos , Análise em Microsséries/normas , Análise em Microsséries , Estudos Retrospectivos , Análise em Microsséries/instrumentação , Análise em Microsséries/estatística & dados numéricos
16.
J Med Virol ; 89(8): 1395-1403, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28229467

RESUMO

Coxsackievirus A6 (CV-A6) is an enterovirus, which is known to cause herpangina. However, since 2009 it has frequently been isolated from children with hand, foot, and mouth disease (HFMD). In Japan, CV-A6 has been linked to HFMD outbreaks in 2011 and 2013. In this study, the full-length genome sequencing of CV-A6 strains were analyzed to identify the association with clinical manifestations. Five thousand six hundred and twelve children with suspected enterovirus infection (0-17 years old) between 1999 and 2013 in Hyogo Prefecture, Japan, were enrolled. Enterovirus infection was confirmed with reverse transcriptase-PCR in 753 children (791 samples), 127 of whom (133 samples) were positive for CV-A6 based on the direct sequencing of the VP4 region. The complete genomes of CV-A6 from 22 positive patients with different clinical manifestations were investigated. A phylogenetic analysis divided these 22 strains into two clusters based on the VP1 region; cluster I contained strains collected in 1999-2009 and mostly related to herpangina, and cluster II contained strains collected in 2011-2013 and related to HFMD outbreak. Based on the full-length polyprotein analysis, the amino acid differences between the strains in cluster I and II were 97.7 ± 0.28%. Amino acid differences were detected in 17 positions within the polyprotein. Strains collected in 1999-2009 and those in 2011-2013 were separately clustered by phylogenetic analysis based on 5'UTR and 3Dpol region, as well as VP1 region. In conclusion, HFMD outbreaks by CV-A6 were recently frequent in Japan and the accumulation of genomic change might be associated with the clinical course.


Assuntos
Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus/classificação , Enterovirus/isolamento & purificação , Genoma Viral , Genótipo , Análise de Sequência de DNA , Adolescente , Criança , Pré-Escolar , Análise por Conglomerados , Enterovirus/genética , Feminino , Humanos , Lactente , Japão , Masculino , Epidemiologia Molecular , Filogenia
17.
Sci Rep ; 7: 41485, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28148910

RESUMO

CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect.


Assuntos
Infecções por Coxsackievirus/patologia , Enterovirus Humano B/fisiologia , Inteínas , Miocardite/patologia , Miocardite/virologia , Proteínas Virais de Fusão/metabolismo , Animais , Diferenciação Celular , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/virologia , Ciclização , Células Dendríticas/metabolismo , Meia-Vida , Células HeLa , Humanos , Imunidade Celular , Imunidade Humoral , Masculino , Camundongos Endogâmicos BALB C , Miocardite/imunologia , Reprodutibilidade dos Testes , Trans-Splicing/genética , Vacinação , Proteínas Virais de Fusão/isolamento & purificação
18.
Biochem Biophys Res Commun ; 484(3): 550-556, 2017 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-28131843

RESUMO

Viral myocarditis (VMC) is closely related to apoptosis, oxidative stress, innate immunity, and energy metabolism, which are all linked to mitochondrial dysfunction. A close nexus between mitochondrial dynamics and cardiovascular disease with mitochondrial dysfunction has been deeply researched, but there is still no relevant report in viral myocarditis. In this study, we aimed to explore the role of Dynamin-related protein 1 (Drp1)-linked mitochondrial fission in VMC. Mice were inoculated with the Coxsackievirus B3 (CVB3) and treated with mdivi1 (a Drp1 inhibitor). Protein expression of Drp1 was increased in mitochondria while decreased in cytoplasm and accompanied by excessive mitochondrial fission in VMC mice. In addition, midivi1 treatment attenuate inflammatory cells infiltration in myocardium of the mice, serum Cardiac troponin I (CTnI) and Creatine kinase-MB (CK-MB) level. Mdivi1 also could improved the survival rate of mice and mitochondrial dysfunction reflected as the up-regulated mitochondrial marker enzymatic activities of succinate dehydrogenase (SDH), cytochrome c oxidase (COX) and mitochondrial membrane potential (MMP). At the same time, mdivi1 rescued the body weight loss, myocardial injury and apoptosis of cardiomyocyte. Furthermore, decease in LVEDs and increase in EF and FS were detected by echocardiogram, which indicated the improved myocardial function. Thus, Drp1-linked excessive mitochondrial fission contributed to VMC and midivi1 may be a potential therapeutic approach.


Assuntos
Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B , Mitocôndrias/metabolismo , Miocardite/metabolismo , Miocardite/patologia , Miocárdio/patologia , Animais , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Regulação para Baixo , Coração/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/patologia , Miocardite/virologia
19.
Virol J ; 13(1): 168, 2016 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-27724948

RESUMO

BACKGROUND: Viral myocarditis, which is often caused by coxsackievirus B3 (CVB3), is a serious clinical disorder characterized by excessive myocardial inflammation. Valproic acid (VPA) is described as a histone deacetylase inhibitor that has anti-inflammatory effects in several inflammatory diseases. However, the role and the detailed mechanism of VPA in viral myocarditis remain unclear. METHODS: Experimental CVB3-induced myocarditis was induced in mice by intraperitoneally (i.p.) infected with CVB3. VPA was i.p. administered from day 0 to day 7. The survival, body weight loss, and myocarditis severity of mice were recorded. Th17 and Treg cells in spleen were analyzed by flow cytometry. Th17/Treg cell-related cytokine expressions were quantified by ELISA. The effect of VPA on Th17 and Treg cells differentiation was examined in vitro and in vivo. RESULTS: Administration of VPA significantly attenuated the clinical severity of myocarditis, and the overall mortality from CVB3-induced myocarditis. The infiltration of Th17 and Treg cells, as well as the serum level of related cytokines (IL-17A and IL-10), were increased in CVB3 infected mice. In addition, VPA decreased the percentage of splenic Th17 cells while increased the percentage of Treg cells. Moreover, VPA downregulated the expression of IL-17A and upregulated IL-10 in serum and heart tissues of CVB3 infected mice. Additionally, VPA directly inhibited the differentiation of Th17 cells and promoted both the differentiation and suppressive function of Treg cells in vitro and in vivo. CONCLUSIONS: Our results suggest that VPA may thus be a promising strategy in the therapy of viral myocarditis.


Assuntos
Infecções por Coxsackievirus/tratamento farmacológico , Enterovirus Humano B/isolamento & purificação , Fatores Imunológicos/administração & dosagem , Miocardite/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Ácido Valproico/administração & dosagem , Animais , Peso Corporal , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Citocinas/análise , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos BALB C , Miocardite/patologia , Miocardite/virologia , Baço/imunologia , Análise de Sobrevida , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento
20.
PLoS One ; 11(8): e0159971, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27486657

RESUMO

OBJECTIVES: To address the question as to whether echocardiographic and/or microcomputed tomography (microCT) analysis can be utilized to assess the extent of Coxsackie B virus (CVB) induced myocarditis in the absence of left ventricular dysfunction in the mouse. BACKGROUND: Viral myocarditis is a significant clinical problem with associated inflammation of the myocardium and myocardial injury. Murine models of myocarditis are commonly used to study the pathophysiology of the disease, but methods for imaging the mouse myocardium have been limited to echocardiographic assessment of ventricular dysfunction and, to a lesser extent, MRI imaging. METHODS: Using a murine model of myocarditis, we used both echocardiography and microCT to assess the extent of myocardial involvement in murine myocarditis using both wild-type mice and CVB cleavage-resistant dystrophin knock-in mice. RESULTS: Areas of increased echogenicity were only observed in the myocardium of Coxsackie B virus infected mice. These echocardiographic abnormalities correlated with the extent of von Kossa staining (a marker of membrane permeability), inflammation, and fibrosis. Given that calcium phosphate uptake as imaged by von Kossa staining might also be visualized using microCT, we utilized microCT imaging which allowed for high-resolution, 3-dimensional images of radiodensities that likely represent calcium phosphate uptake. As with echocardiography, only mice infected with Coxsackie B virus displayed abnormal accumulation of calcium within individual myocytes indicating increased membrane permeability only upon exposure to virus. CONCLUSIONS: These studies demonstrate new, quantitative, and semi-quantitative imaging approaches for the assessment of myocardial involvement in the setting of viral myocarditis in the commonly utilized mouse model of viral myocarditis.


Assuntos
Infecções por Coxsackievirus/complicações , Infecções por Coxsackievirus/diagnóstico , Ecocardiografia , Miocardite/diagnóstico , Miocardite/virologia , Miocárdio/patologia , Microtomografia por Raio-X , Animais , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Distrofina/genética , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miocardite/genética , Miocardite/patologia , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/virologia
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