RESUMO
N,N'-bisheteryl derivatives of dispirotripiperazine (DSTP) are a novel class of antiviral compounds with some of their representatives very effectively inhibiting the replication of herpes simplex virus type 1 (HSV-1) in cell culture. Using one representative of these compounds, the N,N'-bis(1-oxido[1,2,5]oxadiazolo[3,4-d]pyrimidin-7-yl)-3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (DSTP 27), we here further tried to elucidate the molecular mechanisms responsible for the antiviral activity. The results from plaque reduction assays under a variety of conditions suggest that inhibition of HSV-1 strain Kupka replication by DSTP 27 occurs at the level of viral attachment by blockade of heparan sulfate (HS) structures on the cell surface that are used as viral receptors. In contrast to heparin and pentosan polysulfate, pretreatment of cells with DSTP 27 resulted in efficient inhibition of viral adsorption and replication persisting several hours after removal of the inhibitor. Specific binding of DSTP 27 to heparin was demonstrated in vitro. Titrations of gC-positive and gC-negative pseudorabies virus (PrV) mutants on HS-positive and HS-negative cell lines confirmed that inhibitory action of DSTP 27 is strictly HS dependent. Aside from HSV-1 Kupka and PrV, DSTP 27 efficiently inhibits growth of several HSV-1 and HSV-2 strains, among them aciclovir/foscarnet-resistant strains, human cytomegalovirus, human respiratory syncytial virus, and human immunodeficiency viruses known to attach to the cell surface via HS.
Assuntos
Antivirais/farmacologia , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Suídeo 1/efeitos dos fármacos , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Receptores Virais/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Heparitina Sulfato/química , Herpesvirus Humano 1/patogenicidade , Herpesvirus Suídeo 1/genética , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Mutação , Oxidiazóis/metabolismo , Pirimidinas/metabolismo , Receptores Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
During the search for new antivirals, various N,N'-bis-5-pyrimidyl derivatives of 3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (dispirotripiperazine) were synthesized. To reveal relationships between chemical structure and antiviral activity, the compounds were characterized by fast atom bombardment mass, nuclear magnetic resonance, infra red spectroscopy, and elemental analysis and examined for cytotoxicity, inhibition of cell growth and antiviral activity under in vitro conditions. The results of this study demonstrate an excellent compatibility of the test compounds for confluent as well as proliferating cells and a potent structure-dependent inhibition of herpes simplex virus type 1 replication when added during viral adsorption. Functional group analysis revealed that both the dispirotripiperazine as well as the pyrimidine ring with a nitro group in the 5 position are necessary for activity. A reduction of electron density in the terminal pyrimidine rings enhanced the antiviral activity whereas electron donor substitutions reduced it. Introduction of a methyl group in position 2 of the pyrimidine had no influence on cytotoxicity or antiviral activity.
Assuntos
Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Piperazinas/farmacologia , Compostos de Espiro/farmacologia , Animais , Antivirais/síntese química , Antivirais/toxicidade , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Células HeLa , Herpesvirus Humano 1/fisiologia , Humanos , Piperazinas/síntese química , Piperazinas/toxicidade , Compostos de Espiro/síntese química , Compostos de Espiro/toxicidade , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Apoptotic cardiac myocytes have been described in chronic heart failure, but no data exist on the relationship between these 'damaged' myocytes and myocardial detection of enterovirus RNA often associated with dilated cardiomyopathy (DCM). DESIGN: In patients with idiopathic DCM, endomyocardial biopsy samples were studied for enteroviral RNA by one step reverse transcription-polymerase chain reaction (PCR) and a subsequent hybridization of the PCR product using a Southern blot technique. The endomyocardial biopsies were further investigated for markers of cell damage and apoptosis: DNA fragmentation and expression of tissue-transglutaminase (TTG) in the myocytes using the in-situ endlabelling method or an anti-TTG-staining, respectively. To assess the prognostic significance of these two markers the correlation between the percentage of myocytes positive both for DNA fragmentation and TTG (the index of damaged myocytes) and the hemodynamic course of the patients during a mean follow-up period of 15.9 +/- 6.2 months was investigated prospectively by echocardiography. RESULTS: In 14 (45%) of the 31 patients with idiopathic DCM, enteroviral RNA was found in the endomyocardial biopsy samples, while 17 patients (55%) were enterovirus-negative. In enterovirus-positive patients, the index of 'damaged' myocytes was significantly lower (10.7 +/- 4.9% vs. 19.2 +/- 8.8%, P = 0.002) and the left ventricular ejection fraction (LVEF) improved significantly (P = 0.00017 vs. P = 0.13) during long-term follow-up. In addition, a weak negative correlation was seen between the index of damaged myocytes and the changes in LVEF in all patients during long-term follow-up (r = - 0.48, P = 0.004). CONCLUSION: Our results favour the view that enterovirus-positive patients with DCM have less damaged myocytes and a better haemodynamic course than enterovirus-negative patients.
Assuntos
Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/virologia , Infecções por Enterovirus/patologia , Enterovirus/isolamento & purificação , Proteínas de Ligação ao GTP/análise , Transglutaminases/análise , Adulto , Biópsia , Cardiomiopatia Dilatada/metabolismo , Fragmentação do DNA , Enterovirus/genética , Infecções por Enterovirus/metabolismo , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Valor Preditivo dos Testes , Prognóstico , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Viral/análiseRESUMO
Infections with coxsackievirus B3 (CVB3) are common causes of myocarditis in humans. One detail of CVB3-induced pathogenesis is apoptosis. The interaction between the capsid protein VP2 of the myocardial virus variant CVB3H3 and the proapoptotic host cell protein Siva has recently been observed. In order to characterize the interaction between both proteins more precisely, the binding activity of the CVB3H3 VP2 to Siva was compared to that of the mutant virus CVB3H310A1 VP2. We found that the asparagine at position 165 in VP2 is essential for a stable interaction with Siva influencing also the induction of apoptosis, viral spread, and inflammatory responses in vivo. Furthermore, the specific binding site of Siva to VP2 is located at amino acid positions 118-136. Together, these results show that the interaction between VP2 of CVB3H3 and Siva is a highly specific process involving distinct amino acids on both proteins that most likely influence the outcome of CVB3-caused disease.
Assuntos
Apoptose , Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , Enterovirus Humano B/patogenicidade , Infecções por Enterovirus/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Capsídeo/química , Capsídeo/genética , Proteínas do Capsídeo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pâncreas/patologia , Pâncreas/virologia , RNA Viral/análise , RNA Viral/sangue , Transcrição GênicaRESUMO
Coxsackievirus B3 (CVB3)-induced myocarditis in NMRI mice represents a model for studying the pathogenesis of this chronic heart disease. Previously, we reported on specific cytokine patterns during the acute stage of myocarditis since cytokines are thought to play the important role in this cardiomyopathy. In this study, the expression of various cytokine mRNAs and CVB3-RNA kinetics was examined with particular emphasis on the late phase of myocarditis, by using reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). In addition, replicating and persisting CVB3-RNAs were semiquantified by PCR-ELISA. Distinct histopathological changes responsible for ongoing heart disease were found and characterized by increased fibrosis, persistent cellular infiltration and degenerated necrotic myocytes. One of the most important findings of this study was that the mRNA-expression of TNF- alpha, IL-1 alpha, interferon- gamma, IL-10, IL-18, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor- beta (TGF- beta) and inducible nitric oxide synthase (iNOS) persisted as long as 98 days after the virus infection. The induction of IL-10 as well as IFN- gamma mRNAs was also verified by ISH and IHC at days 28 and 98 p.i. The clearly apparent persistence of the viral genomes in the myocardium of infected mice was confirmed by seminested PCR, ISH, and PCR-enzyme linked immunoabsorbent assay (ELISA), showing the highest amount of viral RNA in myocardial cells at day 7 after infection. These data indicate that the persistence of viral RNA is associated with persistently high levels of cytokine mRNAs which, when translated, could severely contribute to pathological changes and injury of connective tissue in the chronic stage of myocarditis.
Assuntos
Infecções por Coxsackievirus/imunologia , Citocinas/biossíntese , Enterovirus Humano B/fisiologia , Miocardite/imunologia , Miocárdio/imunologia , Animais , Doença Crônica , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Citocinas/genética , Enterovirus Humano B/genética , Ensaio de Imunoadsorção Enzimática , Coração/virologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos , Miocardite/induzido quimicamente , Miocardite/patologia , Miocardite/virologia , Miocárdio/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause. Experimental data indicate that cytokines are involved in controlling CVB3 replication. Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (T(H)1)-specific gamma interferon (IFN-gamma) or the T(H)2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established. Coding regions of murine cytokines were cloned into the 5' end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site. Correct processing releases active cytokines, and the concentration of IFN-gamma and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays. In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis. Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection. In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing. Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-gamma)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable. This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/genética , Enterovirus Humano B/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Miocardite/imunologia , Animais , Clonagem Molecular , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Variação Genética , Coração/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miocardite/patologia , Miocardite/virologia , Pâncreas/patologia , Pâncreas/virologia , Proteínas Recombinantes/biossínteseRESUMO
Coxsackievirus B3 (CVB3) causes acute and chronic myocarditis, which is accompanied by an intense mononuclear leukocyte infiltration. Because myocardial tissue damage may either result from viral infections or from a dysregulated immune response, the susceptibility of human monocytes and macrophages to CVB3 was examined in this study with regard to virus replication, virus persistence, and release of cytokines. Monocytes were infected by CVB3 as shown by the intracellular appearance of plus- and minus-strand viral RNA, which was also capable of persisting for more than 10 days. Fresh monocytes were not permissive for full virus replication whereas monocyte-derived macrophages yielded a low amount of new viruses, which led to cell death. Although CVB3 infection induced the mRNA for the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6, only little cytokine production occurred. When infected monocytes were stimulated in addition by lipopolysaccharides (LPS), cytokine production was partially suppressed. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 on the mRNA and the protein level. These data show a dysregulated cytokine response in CVB3-exposed human monocytes and macrophages, which is characterized by a suppression of proinflammatory cytokines and a dominance of IL-10. This viral strategy may aid CVB3, causing chronic myocardiopathy.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus Humano B , Interleucina-10/biossíntese , Monócitos/fisiologia , Morte Celular , Diferenciação Celular , Células Cultivadas , Infecções por Coxsackievirus/virologia , Citocinas/biossíntese , Citocinas/genética , Células HeLa , Humanos , Interleucina-10/genética , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/efeitos dos fármacos , Monócitos/virologia , RNA Mensageiro , RNA Viral/análise , Fatores de Tempo , Replicação ViralRESUMO
In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin-Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.
Assuntos
Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Amantadina , Animais , Divisão Celular , Linhagem Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral , Cães , Enterovirus Humano B/patogenicidade , Foscarnet , Guanidina , Células HeLa , Herpesvirus Humano 1/patogenicidade , Humanos , Vírus da Influenza A/patogenicidade , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , Fatores de Tempo , Ensaio de Placa ViralRESUMO
Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.
Assuntos
Infecções por Enterovirus/veterinária , Enterovirus Suínos/classificação , Enterovirus Suínos/genética , Genoma Viral , Filogenia , Doenças dos Suínos/virologia , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Infecções por Enterovirus/virologia , Enterovirus Suínos/imunologia , Evolução Molecular , Técnica Indireta de Fluorescência para Anticorpo , Dados de Sequência Molecular , Testes de Neutralização , Conformação de Ácido Nucleico , Fenótipo , RNA Ribossômico 18S/genética , RNA Viral/genética , Alinhamento de Sequência , Sorotipagem , SuínosRESUMO
DNA immunizations with the major structural protein VP1 of Coxsackie virus B3 (CVB3) have been previously found to protect BALB/c mice from lethal challenge. Here we report that the other CVB3 capsid proteins, VP2, VP3, and VP4, were less effective at preventing CVB3-caused disease. The application of pCMV/VP1 as a vaccine caused decreased myocyte destruction, reduced viral load in the heart tissue, accelerated antibody induction, and an early cytokine expression in heart tissue. In summary, our results indicate that the induction of B cell and/or T cell memory in vaccinated mice prior to challenge is responsible for the protection observed.
Assuntos
Infecções por Coxsackievirus/prevenção & controle , Enterovirus/imunologia , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antivirais/imunologia , Peso Corporal , Capsídeo/genética , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/virologia , Citocinas/análise , Citocinas/imunologia , Intervalo Livre de Doença , Enterovirus/genética , Vetores Genéticos , Coração/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas de DNA/imunologia , Carga ViralRESUMO
The coxsackievirus B3 (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property, the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VP1 capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and I92 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VP1 are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 PD and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces.
Assuntos
Capsídeo/metabolismo , Enterovirus Humano B/metabolismo , Enterovirus Humano B/patogenicidade , Variação Genética/genética , Substituição de Aminoácidos/genética , Animais , Anticorpos/farmacologia , Sítios de Ligação , Antígenos CD55/metabolismo , Capsídeo/química , Capsídeo/genética , Linhagem Celular , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Efeito Citopatogênico Viral , Análise Mutacional de DNA , DNA Recombinante/genética , Enterovirus Humano B/classificação , Enterovirus Humano B/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Especificidade de Órgãos , Fenótipo , Polimorfismo Genético/genética , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptores Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Porcine enteroviruses (PEV) comprising at least 13 serotypes grouped into three species are described as causative agents of neurological disorders, fertility disorders, and dermal lesions of swine. Despite their well-documented acid stability, enteric infection route, and similarity of clinical symptoms, most of the porcine enterovirus (PEV) serotypes are set apart from the genus Enterovirus of the Picornaviridae. Hence, PCR procedures used commonly to detect enteroviruses are not applicable to epizootic relevant PEV serotypes. A nested RT-PCR protocol is described now suited to detect all known porcine enterovirus serotypes using three sets of primer pairs. These primer pairs were designed to amplify either highly conserved sequences of the 5'-nontranslated region (5'-NTR) or the polymerase gene region of the relevant virus species. All 13 acknowledged serotypes of three PEV species and several field isolates of clinical specimens were detectable. The specificity of the PCR procedure is supported by the observation that RT-PCR-positive field isolates coincide with serological PEV classification. PEV PCR is more rapid and less laborious than the time-consuming virus isolation by tissue culture techniques over several passages and serotyping. Because other viruses such as classical swine fever virus, pseudorabies virus, porcine parvovirus, swine vesicular disease virus, and foot-and-mouth disease virus may cause diseases with similar clinical symptoms, PCR detection of all PEVs closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the diagnosis of all relevant viruses causing such symptoms.
Assuntos
Infecções por Enterovirus/veterinária , Enterovirus Suínos/isolamento & purificação , RNA Viral/isolamento & purificação , Doenças dos Suínos/virologia , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Efeito Citopatogênico Viral , Primers do DNA , RNA Polimerases Dirigidas por DNA/genética , Infecções por Enterovirus/virologia , Enterovirus Suínos/classificação , Enterovirus Suínos/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência , Sorotipagem , SuínosRESUMO
Mice develop a marked age-related susceptibility to myocardial coxsackievirus B3 (CVB3) infections. The lesions observed in mice resemble closely those seen in the human disease. Experimental murine models of CVB3-induced myocarditis have shown that both, host and viral genetic factors, can influence susceptibility to the infection as well as the persistence and progression of the disease. Recently, we have shown that CD4 T cell-deficient MHC Class II knockout mice develop a strong fibrosis with virus persistence in the heart tissue and without production of neutralizing antibodies. To examine the role of CD4+ T cells and especially the role of the T helper 1 cell response for the outcome and pathogenesis of CVB3-induced myocarditis in more detail, 2 different mouse strains with identical genetic background (H-2b) were infected with CVB3-Mü/J (Nancy strain). Immunocompetent C57BL/6 mice and mice with targeted disruption of interleukin (IL-)4 gene (IL-4-/- mice) developed a severe acute myocarditis on day 7 post infection (p.i.). The CVB3-induced inflammation was cured until the 21st day p.i. in hearts of C57BL/6 mice. IL-4-/- mice with insufficient T helper-2 cell immune response developed a severe myocardial damage between day 7 and 21 p.i. with prolonged virus persistence in the heart tissue. Therefore, we suggest that despite an obvious normal T helper-1 cell cytokine pattern, IL-4-/- mice are more susceptible to long-term heart muscle injuries after infection with CVB3.
Assuntos
Cardiomiopatia Dilatada/imunologia , Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/imunologia , Imunidade Celular/imunologia , Miocardite/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Infecções por Coxsackievirus/diagnóstico , Modelos Animais de Doenças , Humanos , Interleucina-4/genética , Interleucina-4/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/diagnóstico , Miocardite/patologia , Miocárdio/imunologia , Prognóstico , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection [p.i.]). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.
Assuntos
Infecções por Coxsackievirus/enzimologia , Enterovirus Humano B , Miocardite/enzimologia , Óxido Nítrico Sintase/genética , Animais , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/genética , Indução Enzimática/genética , Regulação Viral da Expressão Gênica/genética , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Camundongos , Miocardite/genética , Miocardite/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Óxido Nítrico/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Since cytokines play an important role in the pathogenesis of virus-induced chronic heart diseases, cytokine mRNA expression was studied in coxsackievirus B3-infected NMRI mice during the acute phase of myocarditis until the onset of chronic cardiac disease. Virus replication, cytokine induction, inflammatory cell infiltration and myocardial damage were studied by titer determination, reverse transcription-polymerase chain reaction (RT-PCR), and histopathology. To investigate whether the coxsackievirus B3-induced cytokine mRNA accumulation was only limited to the heart or generalized, spleen and thymus specimens were also included. Surprisingly, interleukin (IL)-10 as a deactivator of T cell and macrophage functions was transcribed in the myocardium nearly in parallel with virus replication from Day 1 through Day 14. At Day 3 p.i., the mRNA of IL-1alpha, tumor necrosis factor (TNF)-alpha, IL-6, and interferon (IFN)-beta accumulated. At Days 4, 7, and 14, IL-12-specific mRNA was produced. Furthermore, increasing amounts of IFN-gamma mRNA were found, whereas IL-2 and IL-4 mRNA remained undetectable. TNF-alpha, IL-1alpha, IL-10, IL-12, and IFN-gamma mRNA persisted into the late stage of myocarditis. In the spleen a closely correlated expression of virus and IL-10-specific mRNAs was also found, and in addition, IFN-beta, TNF-alpha, and IL-6 were detected. In striking contrast to heart and spleen tissue, the distinct expression of viral RNA in the thymus was not accompanied by an increased cytokine mRNA production. These data provide evidence for a unique coxsackievirus B3-induced cytokine pattern in the myocardium and spleen and suggest that persistently expressed IL-10 may play a leading role in acute and chronic myocarditis by subverting the immune response.
Assuntos
Infecções por Coxsackievirus/imunologia , Citocinas/análise , Enterovirus , Miocardite/imunologia , Miocárdio/imunologia , Baço/imunologia , Timo/imunologia , Doença Aguda , Animais , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Enterovirus/isolamento & purificação , Coração/virologia , Interferon beta/análise , Interferon gama/análise , Interleucinas/análise , Masculino , Camundongos , Miocardite/patologia , Miocardite/virologia , Miocárdio/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.
Assuntos
Antígenos CD , Apoptose , Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Ligante CD27 , Capsídeo/genética , Proteínas do Capsídeo , Proteínas de Transporte/genética , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Distribuição Tecidual , Transcrição Gênica , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in immunocompetent C57BL/6 and MHC class II knockout mice with identical genetic backgrounds. STUDY DESIGN/METHODS: We analyzed the histology and immunohistology of myocardial injury, the replicating virus titer, and antibody response in the early and late phase of disease. RESULTS: CVB3-infected C57BL/6 mice showed acute myocarditis, with spontaneous healing, virus elimination, anti-CVB3 IgM/IgG production, and neutralizing antibody response. In contrast, MHC class II knockout mice developed less severe acute myocarditis, persistence of infiltrations and strong fibrosis, virus persistence, and weak IgG response, with absence of virus neutralizing antibodies. CONCLUSIONS: Immunodeficient organisms are more susceptible to long-term heart muscle injuries after infection with CVB3. The presence of CD4+ T cells are necessary to prevent the development of chronic disease.
Assuntos
Infecções por Coxsackievirus/virologia , Enterovirus Humano B , Antígenos de Histocompatibilidade Classe II/imunologia , Miocardite/virologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos CD/análise , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/crescimento & desenvolvimento , Enterovirus Humano B/imunologia , Deleção de Genes , Coração/virologia , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe II/genética , Técnicas Imunológicas , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Miocardite/imunologia , Miocardite/metabolismo , Miocardite/patologia , Miocárdio/química , Miocárdio/imunologia , Miocárdio/patologia , Linfócitos T/química , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/análise , Carga ViralRESUMO
OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population. STUDY DESIGN/METHODS: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA. RESULTS: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection. CONCLUSIONS: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.
Assuntos
Infecções por Coxsackievirus , Modelos Animais de Doenças , Enterovirus Humano B , Miocardite/virologia , Animais , Anticorpos Antivirais/sangue , Doença Crônica , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/isolamento & purificação , Enterovirus Humano B/fisiologia , Coração/virologia , Humanos , Masculino , Camundongos , Miocardite/patologia , Miocárdio/patologia , Pâncreas/virologia , RNA Viral/análise , Replicação ViralRESUMO
The goal of this work was to examine the effect of triple helix-forming oligonucleotides on a gyrase target region and on the activity of the enzyme. Using melting temperature measurements and gel mobility shift analysis, it was found that modified oligonucleotides can form a triple helix along the 29-nucleotide region of a 32-bp duplex representing part of the gyrase DNA-target sequence of the 162-bp fragment from pBR322. Triplex formation with this target region has been achieved at pH 7.5 by using a synthetic oligonucleotide in which cytosine was replaced by the C-nucleoside of 2-aminopyridine. The results of the enzymic experiments in vitro with the 162-bp fragment demonstrated that the cleavage reaction mediated by gyrase can be efficiently inhibited by the triplex-forming oligonucleotide modified with 2-aminopyridine. A possible inhibitory mechanism is discussed.