RESUMO
Intestinal microbiomes are of vital importance in antagonizing systemic viral infection. However, very little literature has shown whether commensal bacteria play a crucial role in protecting against enteric virus systemic infection from the aspect of modulating host innate immunity. In the present study, we utilized an enteric virus, encephalomyocarditis virus (EMCV), to inoculate mice treated with phosphate-buffered saline (PBS) or given an antibiotic cocktail (Abx) orally or intraperitoneally to examine the impact of microbiota depletion on virulence and viral replication in vivo Microbiota depletion exacerbated the mortality, neuropathogenesis, viremia, and viral burden in brains following EMCV infection. Furthermore, Abx-treated mice exhibited severely diminished mononuclear phagocyte activation and impaired type I interferon (IFN) production and expression of IFN-stimulated genes (ISG) in peripheral blood mononuclear cells (PBMC), spleens, and brains. With the help of fecal bacterial 16S rRNA sequencing of PBS- and Abx-treated mice, we identified a single commensal bacterium, Blautia coccoides, that can restore mononuclear phagocyte- and IFNAR (IFN-α/ß receptor)-dependent type I IFN responses to restrict systemic enteric virus infection. These findings may provide insight into the development of novel therapeutics for preventing enteric virus infection or possibly alleviating clinical diseases by activating host systemic innate immune responses via respective probiotic treatment using B. coccoidesIMPORTANCE While cumulative data indicate that indigenous commensal bacteria can facilitate enteric virus infection, little is known regarding whether intestinal microbes have a protective role in antagonizing enteric systemic infection by modulating host innate immunity. Although accumulating literature has pointed out that the microbiota has a fundamental impact on host systemic antiviral innate immune responses mediated by type I interferon (IFN), only a few specific commensal bacteria species have been revealed to be capable of regulating IFN-I and ISG expression, not to mention the underlying mechanisms. Thus, it is important to understand the cross talk between microbiota and host anti-enteric virus innate immune responses and characterize the specific bacterial species that possess protective functions. Our study demonstrates how fundamental innate immune mediators such as mononuclear phagocytes and type I IFN are regulated by commensal bacteria to antagonize enteric virus systemic infection. In particular, we have identified a novel commensal bacterium, Blautia coccoides, that can restrict enteric virus replication and neuropathogenesis by activating IFN-I and ISG responses in mononuclear phagocytes via an IFNAR- and STAT1-mediated signaling pathway.
Assuntos
Infecções por Cardiovirus/prevenção & controle , Vírus da Encefalomiocardite/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade Inata , Interferon Tipo I/imunologia , Viremia/imunologia , Viremia/prevenção & controle , Animais , Antibacterianos/administração & dosagem , Infecções por Cardiovirus/imunologia , Clostridiales/imunologia , Vírus da Encefalomiocardite/patogenicidade , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Simbiose/imunologia , Replicação Viral/imunologiaRESUMO
Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 µL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalomiocardite/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Anticorpos Antivirais/isolamento & purificação , Infecções por Cardiovirus/prevenção & controle , Clonagem Molecular , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Resultado do TratamentoRESUMO
UNLABELLED: Mengovirus, a member of thePicornaviridaefamily, has a broad cell tropism and can cause encephalitis and myocarditis in multiple mammalian species. Attenuation has been achieved by shortening the polycytidine tract in the 5' noncoding region (NCR). A poly(C)-truncated strain of mengovirus, vMC24, resulted in significant tumor regression in immunocompetent BALB/c mice bearing syngeneic MPC-11 plasmacytomas, but the associated toxicities were unacceptable. To enhance its safety profile, microRNA target sequences complementary to miR-124 or miR-125 (enriched in nervous tissue), miR-133 and miR-208 (enriched in cardiac tissue), or miR-142 (control; enriched in hematopoietic tissues) were inserted into the vMC24NCRs. The microRNA-detargeted viruses showed reduced replication and cell killing specifically in cells expressing the cognate microRNAs, but certain insertions additionally were associated with nonspecific suppression of viral fitnessin vivo. In vivotoxicity testing confirmed that miR-124 targets within the 5' NCR suppressed virus replication in the central nervous system while miR-133 and miR-208 targets in the 3' NCR suppressed viral replication in cardiac tissue. A dual-detargeted virus named vMC24-NC, with miR-124 targets in the 5' NCR and miR-133 plus miR-208 targets in the 3' NCR, showed the suppression of replication in both nervous and cardiac tissues but retained full oncolytic potency when administered by intratumoral (10(6)50% tissue culture infectious doses [TCID50]) or intravenous (10(7)to 10(8)TCID50) injection into BALB/c mice bearing MPC-11 plasmacytomas. Overall survival of vMC24-NC-treated tumor-bearing mice was significantly improved compared to that of nontreated mice. MicroRNA-detargeted mengoviruses offer a promising oncolytic virotherapy platform that merits further development for clinical translation. IMPORTANCE: The clinical potential of oncolytic virotherapy for cancer treatment has been well demonstrated, justifying the continued development of novel oncolytic viruses with enhanced potency. Here, we introduce mengovirus as a novel oncolytic agent. Mengovirus is appealing as an oncolytic virotherapy platform because of its small size, simple genome structure, rapid replication cycle, and broad cell/species tropism. However, mengovirus can cause encephalomyelitis and myocarditis. It can be partially attenuated by shortening the poly(C) tract in the 5' NCR but remains capable of damaging cardiac and nervous tissue. Here, we further enhanced the safety profile of a poly(C)-truncated mengovirus by incorporating muscle- and neuron-specific microRNA target sequences into the viral genome. This dual-detargeted virus has reduced pathogenesis but retained potent oncolytic activity. Our data show that microRNA targeting can be used to further increase the safety of an attenuated mengovirus, providing a basis for its development as an oncolytic platform.
Assuntos
Mengovirus , MicroRNAs/genética , Mieloma Múltiplo/terapia , Terapia Viral Oncolítica , Animais , Infecções por Cardiovirus/etiologia , Infecções por Cardiovirus/prevenção & controle , Linhagem Celular , Efeito Citopatogênico Viral , Feminino , Marcação de Genes , Instabilidade Genômica , Humanos , Hospedeiro Imunocomprometido , Mengovirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/imunologia , Síndromes Neurotóxicas/prevenção & controle , Síndromes Neurotóxicas/virologia , Terapia Viral Oncolítica/efeitos adversos , RNA não Traduzido/genética , Replicação ViralRESUMO
BACKGROUND: A photoactive hydrophobic agent 1,5-iodonaphthyl-azide (INA), has been previously shown to completely inactivate the enveloped viruses. INA sequesters into the lipid bilayer of the virus envelope and upon UV-irradiation bind to the hydrophobic domains of the envelope glycoproteins. In our earlier study, we have shown that the Venezuelan equine encephalitis virus (VEEV) genomic RNA was also inactivated during the inactivation of the virus with INA. FINDINGS: In the present study, we evaluated if the RNA inactivation property of INA can be used to inactivate non-enveloped RNA viruses. Encephalomyocarditis virus (EMCV) was used as a model non-enveloped virus. Treatment with INA followed by UV-irradiation resulted in complete inactivation of EMCV. RNA isolated from INA-inactivated EMCV was non-infectious and INA was found to be associated with the viral RNA genome. INA-inactivated EMCV induced robust total antibody response. However binding capacity of INA-inactivated EMCV to neutralizing antibody was inhibited. CONCLUSION: This is the first study to show that INA can completely inactivate non-enveloped virus. Our results suggest that the amino acid composition of the neutralizing epitope may interfere with the protective antibody response generated by the INA-inactivated non-enveloped virus.
Assuntos
Anticorpos Antivirais/biossíntese , Antivirais/farmacologia , Azidas/farmacologia , Infecções por Cardiovirus/prevenção & controle , Vacinas Virais/administração & dosagem , Inativação de Vírus/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/biossíntese , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/virologia , Linhagem Celular , Vírus da Encefalomiocardite/imunologia , Epitopos/química , Epitopos/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/efeitos da radiação , Fibroblastos/virologia , Imunização , Camundongos , Processos Fotoquímicos , RNA Viral/antagonistas & inibidores , Raios Ultravioleta , Vacinas Atenuadas , Inativação de Vírus/efeitos da radiaçãoRESUMO
BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3(+) CD4(+) regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. METHODS: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3(+) Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). RESULTS: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. CONCLUSION: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.
Assuntos
Infecções por Cardiovirus/imunologia , Resistência à Doença/imunologia , Encefalite Viral/imunologia , Linfócitos T Reguladores/fisiologia , Theilovirus/imunologia , Doença Aguda , Animais , Infecções por Cardiovirus/prevenção & controle , Encefalite Viral/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
To evaluate a novel vaccine for porcine encephalomyocarditis virus (EMCV), which causes reproductive failure in pregnant sows, virus like particles (VLPs) were generated and immunized twice in 2 week intervals before sow mating. Sows were divided into 4 groups (n=4, per group). Group 1 was immunized with the alum adjuvant alone, Group 2 with VLPs alone, Group 3 with VLPs mixed alum adjuvant, and Group 4 with a commercial killed vaccine. In Group 2, seroconversion was observed at very low levels, while in Group 3, neutralizing antibodies were maintained at a high level until 30 days after farrowing. Similar levels neutralizing antibodies were observed in Group 4. The gestation period of the pregnant sows was on average 115 days, and no injection site reaction or side effects were observed. The mean temperature of the sows after immunization increased temporarily to 38.7-39.1 °C for 1 day. The numbers and weights of surviving piglets were similar among the groups. These data describe a novel EMCV vaccine composed of VLPs mixed with an alum adjuvant that is safe to use during sow gestation and induces and maintains high levels of seroconversion. This vaccine could thus be a candidate for protecting against EMCV induced reproductive failure in pig farms.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen , Infecções por Cardiovirus/veterinária , Vírus da Encefalomiocardite/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Infecções por Cardiovirus/prevenção & controle , Infecções por Cardiovirus/virologia , Feminino , Gravidez , Suínos , Doenças dos Suínos/virologiaRESUMO
Encephalomyocarditis virus (EMCV) could infect many host species and cause acute myocarditis and sudden death in pre-weaned piglets. It was necessary to develop new antiviral strategies for the treatment of the virus infection. Here, four plasmids expressing shRNA (small hairpin RNA) targeted to 1D or 3AB protein genes of EMCV were constructed and their inhibition efficiency on the replication of EMCV was evaluated in both BHK21 cells and mice. The results showed that three out of those four shRNA constructs could significantly inhibit EMCV replication in BHK21 cells on the levels of viral RNA and protein. Moreover, it was found that the shRNAs could suppress significantly the load of EMCV in the brain tissue of the mice pretreated with the constructs for 6-24 h. The clinical signs and pathological lesions of the mice in the groups inoculated with the shRNA constructed were milder obviously, compared with those in pSUPER-mN3 and challenge control groups. The survival rates of mice inoculated with pSUPER-3AB-1, pSUPER-3AB-2, and pSUPER-1D-1 for 12 h was 100, 80, and 40%, respectively, while, in the control groups it was only 20%. It indicated that the vector-based shRNA targeting to 3AB and 1D genes might be a potential anti-EMCV strategy.
Assuntos
Antivirais/metabolismo , Infecções por Cardiovirus/prevenção & controle , Vírus da Encefalomiocardite/crescimento & desenvolvimento , Genes Virais , RNA Interferente Pequeno/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Encéfalo/virologia , Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/virologia , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Vírus da Encefalomiocardite/genética , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Análise de Sequência de DNA , Análise de Sobrevida , Resultado do Tratamento , Carga ViralRESUMO
Infection of dendritic and glial cells with Theiler's murine encephalomyelitis virus (TMEV) induces various cytokines via Toll-like receptor- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways. However, the involvement and role of MDA5 in cytokine gene activation and the pathogenesis of TMEV-induced demyelinating disease are largely unknown. In this study, we demonstrate that MDA5 plays a critical role in the production of TMEV-induced alpha interferon (IFN-α) during early viral infection and in protection against the development of virus-induced demyelinating disease. Our results indicate that MDA5-deficient 129SvJ mice display significantly higher viral loads and apparent demyelinating lesions in the central nerve system (CNS) accompanied by clinical symptoms compared with wild-type 129SvJ mice. During acute viral infection, MDA5-deficient mice produced elevated levels of chemokines, consistent with increased cellular infiltration, but reduced levels of IFN-α, known to control T cell responses and cellular infiltration. Additional studies with isolated CNS glial cells from these mice suggest that cells from MDA5-deficient mice are severely compromised in the production of IFN-α upon viral infection, which results in increased cellular infiltration and viral loads in the CNS. Despite inadequate stimulation, the overall T cell responses to the viral determinants were significantly elevated in MDA5-deficient mice, reflecting the increased cellular infiltration. Therefore, the lack of MDA5-mediated IFN-α production may facilitate a massive viral load and elevated cellular infiltration in the CNS during early viral infection, leading to the pathogenesis of demyelinating disease.
Assuntos
Infecções por Cardiovirus/prevenção & controle , RNA Helicases DEAD-box/genética , Theilovirus/isolamento & purificação , Animais , Sequência de Bases , Infecções por Cardiovirus/virologia , Primers do DNA , Helicase IFIH1 Induzida por Interferon , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Theilovirus/genéticaRESUMO
Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4(+), CD8(+) and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity.
Assuntos
Doenças Desmielinizantes/imunologia , Modelos Animais de Doenças , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Theilovirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/metabolismo , Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/prevenção & controle , Proliferação de Células , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Índice de Gravidade de Doença , Linfócitos T Reguladores/metabolismo , Theilovirus/efeitos dos fármacosRESUMO
Five phosphodipeptides were synthesized; two of them (H-Lys-Ala(P) and H-Pro-Ala(P) had interferon-induced activity. These dipeptides at millimolar concentrations (10(-4)) and 10(-5) M) induced the synthesis of late (40-hour) interferon in human peripheral blood lymphocytes. The dipeptides H-Lys-Ala(P) and H-Pro-Ala(P) showed a protective antiviral activity in in vivo studies when singly intraperitoneally administered to mice 2 hours before inoculation with murine encephalomyocarditis virus.
Assuntos
Antivirais/farmacologia , Dipeptídeos/farmacologia , Indutores de Interferon/farmacologia , Interferons/imunologia , Fosfopeptídeos/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Infecções por Cardiovirus/prevenção & controle , Linhagem Celular , Dipeptídeos/síntese química , Dipeptídeos/química , Relação Dose-Resposta a Droga , Vírus da Encefalomiocardite/efeitos dos fármacos , Humanos , Injeções Intraperitoneais , Indutores de Interferon/síntese química , Indutores de Interferon/química , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Fosfopeptídeos/síntese química , Fosfopeptídeos/químicaRESUMO
Rat theilovirus is a commonly reported infection in research rat colonies. The author's institution experienced an outbreak of rat theilovirus in a breeding colony of unique outbred rats. To manage this outbreak, the institution chose to use a 'test and cull' strategy because this approach is reported to be successful in mouse colonies infected with Theiler's murine encephalomyelitis virus, a related virus. Here the author describes the outbreak and subsequent management of rat theilovirus. The strategy successfully cleared the virus from the rat colony.
Assuntos
Infecções por Cardiovirus/veterinária , Surtos de Doenças/veterinária , Transmissão de Doença Infecciosa/veterinária , Doenças dos Roedores/epidemiologia , Theilovirus/isolamento & purificação , Criação de Animais Domésticos/métodos , Animais , Animais não Endogâmicos , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Infecções por Cardiovirus/epidemiologia , Infecções por Cardiovirus/prevenção & controle , Surtos de Doenças/prevenção & controle , Transmissão de Doença Infecciosa/prevenção & controle , Eutanásia Animal , Feminino , Masculino , Ratos , Doenças dos Roedores/prevenção & controle , Theilovirus/imunologiaRESUMO
Ligation of CD27, a member of the tumor necrosis factor (TNF) receptor family, by its ligand CD70 is thought to be important in T cell activation, expansion and survival, B cell activation, and NK cell activation. We examined the role of CD70 in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) mice. Blocking of CD70 in effector phase by anti-CD70 monoclonal antibody (mAb) suppressed the development of TMEV-IDD. The number of IFN-gamma- or TNF-alpha-producing cells in the spleen and mRNA levels of IFN-gamma and TNF-alpha in spinal cord were decreased in mice treated with anti-CD70 mAb at the effector phase. In contrast, treatment with anti-CD70 mAb in induction phase failed to reduce these responses, compared to nonspecific IgG-treated control mice. These data suggest that CD70 is critically involved in the pathogenesis of TMEV-IDD and that antibodies against CD70 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Ligante CD27/imunologia , Infecções por Cardiovirus/prevenção & controle , Doenças Desmielinizantes/prevenção & controle , Fatores Imunológicos/uso terapêutico , Theilovirus , Animais , Ligante CD27/fisiologia , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/metabolismo , Citocinas/metabolismo , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/metabolismo , Feminino , Interferon gama/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Baço/imunologia , Baço/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: The aim of study is to investigate the effect of nifedipine on viral myocarditis in an animal model. MAIN METHODS: Four-week-old male DBA/2 mice were inoculated with 2 pfu of encephalomyocarditis virus (EMCV) and randomized to nifedipine (n=10) or control (n=10) group. The control group was fed by regular chow and the nifedipine group contained 0.01% of nifedipine. Mast cell density was counted, and expressions of messenger RNAs of stem cell factor (SCF), matrix metalloproteinases (MMPs), pro-collagen I, mast cell proteases, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were evaluated by RT-PCR. KEY FINDINGS: The area of myocardial necrosis was smaller in the nifedipine vs the control group (mean+/-SD, 1.2+/-1.3% vs 3.8+/-1.8%, respectively, P<0.005). The mast cell density (count/mm(2)) was lower in the nifedipine vs the control group (mean+/-SD, 0.23+/-0.16 vs 1.08+/-0.45, respectively, P<0.0005). The expressions of MMPs, mast cell proteases, TNF-alpha, IL-6, SCF and pro-collagen I were lower in the nifedipine group than in the control group (P<0.05). SIGNIFICANCE: Nifedipine inhibited the activation of various participants in inflammatory and immune reactions in EMCV myocarditis.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Infecções por Cardiovirus/prevenção & controle , Vírus da Encefalomiocardite/fisiologia , Miocardite/prevenção & controle , Nifedipino/farmacologia , Animais , Biomarcadores/metabolismo , Infecções por Cardiovirus/metabolismo , Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/virologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/virologia , Interleucina-5/genética , Interleucina-5/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastócitos/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Miocardite/metabolismo , Miocardite/patologia , Miocardite/virologia , Miocárdio/patologia , Necrose/tratamento farmacológico , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Triptases/genética , Triptases/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Encephalomyocarditis virus (EMCV) infection can cause acute myocarditis and sudden death in pre-weaned piglets as well as severe reproductive failure in sows. In this study, two recombinant adenoviruses containing capsid precursor polypeptide P1 alone (Ad-P1) and P1 plus nonstructural protein 2A and 3C protease coding regions (Ad-P12A3C) of EMCV were respectively constructed using replication-defective human adenovirus serotype 5 as vector, and their antibody responses and protective efficacies against a lethal EMCV challenge were evaluated in mice. Both Ad-P1 and Ad-P12A3C were confirmed to be capable of expressing VP1 protein in BHK21 cells by immunoperoxidase monolayer assay (IPMA). The results showed that mice vaccinated once or twice with Ad-P1 and Ad-P12A3C generated specific antibody response against VP1 protein of EMCV. Although Ad-P1 induced higher antibody titers, virus-neutralizing antibody response was considerably less (p<0.05), compared to that of Ad-P12A3C. Upon challenging with a virulent EMCV strain, Ad-P12A3C elicited efficacious protection (100% for both vaccination once and twice) in the vaccinated mice; whereas the mice immunized with Ad-P1 showed a lower protection (12.5% for vaccination once and 75% for twice). Our work suggests that the recombinant adenovirus (Ad-P12A3C) containing the capsid precursor polypeptide coding region (P1) plus nonstructural protein 2A and 3C protease genes have an excellent potential to be used as a vaccine that can provide sufficient protective efficacy against EMCV infection in animals.
Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , Infecções por Cardiovirus/prevenção & controle , Cisteína Endopeptidases/genética , Vírus da Encefalomiocardite/imunologia , Vacinação , Vacinas de DNA/imunologia , Proteínas Virais/genética , Vacinas Virais/imunologia , Proteases Virais 3C , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Linhagem Celular , Feminino , Vetores Genéticos/imunologia , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Precursores de Proteínas/genética , Recombinação Genética , Vacinas de DNA/administração & dosagem , Proteínas não Estruturais Virais/genética , Vacinas Virais/administração & dosagemRESUMO
The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.
Assuntos
Infecções por Cardiovirus/enzimologia , Infecções por Cardiovirus/prevenção & controle , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Infecções por Rhabdoviridae/enzimologia , Infecções por Rhabdoviridae/prevenção & controle , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/fisiologia , Vírus da Encefalomiocardite/imunologia , Feminino , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
We have demonstrated previously that the C-terminal gamma interferon (IFN-gamma) mimetic peptide consisting of residues 95 to 133 [IFN-gamma(95-133)], which contains the crucial IFN-gamma nuclear localization sequence (NLS), has antiviral activity in tissue culture. Here we evaluate the efficacy of this peptide and its derivatives first in vitro and then in an animal model of lethal viral infection with the encephalomyocarditis (EMC) virus. Deletion of the NLS region from the IFN-gamma mimetic peptide IFN-gamma(95-133) resulted in loss of antiviral activity. However, the NLS region does not have antiviral activity in itself. Replacing the NLS region of IFN-gamma(95-133) with the NLS region of the simian virus 40 large T antigen retains the antiviral activity in tissue culture. IFN-gamma(95-133) prevented EMC virus-induced lethality in mice in a dose-dependent manner compared to controls. Mice treated with IFN-gamma(95-133) had no or low EMC virus titers in their internal organs, whereas control mice had consistently high viral titers, especially in the heart tissues. Injection of B8R protein, which is encoded by poxviruses as a defense mechanism to neutralize host IFN-gamma, did not inhibit IFN-gamma(95-133) protection against a lethal dose of EMC virus, whereas mice treated with rat IFN-gamma were not protected. The data presented here show that the IFN-gamma mimetic peptide IFN-gamma(95-133) prevents EMC virus infection in vivo and in vitro and may have potential against other lethal viruses, such as the smallpox virus, which encodes the B8R protein.
Assuntos
Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/prevenção & controle , Vírus da Encefalomiocardite/imunologia , Interferon gama/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/uso terapêutico , Antivirais/toxicidade , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vírus da Encefalomiocardite/efeitos dos fármacos , Interferon gama/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Mimetismo Molecular/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/toxicidade , Peptídeos/toxicidade , Ensaio de Placa ViralRESUMO
Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.
Assuntos
Antivirais/imunologia , Vírus da Encefalomiocardite/imunologia , Herpesvirus Humano 2/imunologia , Interferons/biossíntese , Interferons/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Infecções por Arenaviridae/prevenção & controle , Infecções por Arenaviridae/virologia , Infecções por Cardiovirus/prevenção & controle , Infecções por Cardiovirus/virologia , Linhagem Celular Tumoral , Progressão da Doença , Vírus da Encefalomiocardite/fisiologia , Feminino , Regulação Viral da Expressão Gênica , Herpes Genital/patologia , Herpes Genital/prevenção & controle , Herpes Genital/virologia , Humanos , Interferons/deficiência , Interferons/genética , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vagina/patologia , Vagina/virologiaRESUMO
The occurrence of encephalomyocarditis virus (EMCV) among domestic pigs and wild boar in several European countries is described and discussed. From 1990 to 2001 clinical outbreaks were analysed and serum samples, partly from existing screening programmes, were tested for antibodies against EMCV. Most clinical EMCV outbreaks were reported in Belgium (320), followed by Italy (110), Greece (15) and Cyprus (6). The outbreaks appeared to be clustered in 'endemic areas' with an increase in outbreaks during the autumn and winter months. The within-herd seroprevalence measured in clinically affected pig farms varied considerably among farms (2-87%), with age (0-84%) and by country. Data from farms with no clinical disease showed that subclinical infection with EMCV was found both within (seroprevalence 6-62%) and outside (up to 17 %) the endemic areas of the clinically affected countries as well as in the non-clinically affected countries Austria and France (3-5.4%). Among wild boar, the seroprevalence varied between 0.6 and 10.8%, and a study in Belgium found a prevalence of virus infection of 3.3%.
Assuntos
Infecções por Cardiovirus/veterinária , Surtos de Doenças/veterinária , Vírus da Encefalomiocardite/isolamento & purificação , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/prevenção & controle , Animais , Animais Domésticos , Animais Selvagens , Infecções por Cardiovirus/epidemiologia , Infecções por Cardiovirus/prevenção & controle , Europa (Continente)/epidemiologia , Estações do Ano , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/etiologiaRESUMO
BACKGROUND: Nuclear factor kappa B (NF-kappaB) is activated by several factors, which increase the inflammatory response, and this activation, in turn, leads to the expression of several genes such as cytokines, and may play an important role in cardiovascular diseases. AIMS: The aim of the study is to examine the effect of SUN C8079, a newly synthesized NF-kappaB inhibitor in vitro and in vivo. METHODS: We examined the effects of SUN C8079 on the transcriptional responses of NF-kappaB, on activation of NF-kappaB in electrophoretic mobility shift assay, and on the gene expressions of tumor necrosis factor (TNF)-alpha and iNOS. We also studied effects of SUN C8079 on lethal endotoxemia and viral myocarditis in mice. RESULTS: SUN C8079 inhibited the lipopolysaccharide (LPS)-induced expression of the genes of TNF-alpha and iNOS by inhibiting the activation of NF-kappaB in vitro. SUN C8079 inhibited the systemic release of TNF-alpha and improved mortality in LPS-treated mice. In addition to protecting mice against lethal endotoxemia, SUN C8079 prevented the development of myocarditis due to the encephalomyocarditis virus (EMCV), and inhibited the expressions of proinflammatory cytokines and the iNOS gene in cardiac tissues. CONCLUSION: These findings suggest that the activation of NF-kappaB plays an important role in the pathogenesis of endotoxemia and viral myocarditis, and that the NF-kappaB inhibitor, SUN C8079, may be therapeutic in these disorders.
Assuntos
Endotoxemia/prevenção & controle , Miocardite/prevenção & controle , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Piperidinas/farmacologia , Fator de Necrose Tumoral alfa/genética , Animais , Infecções por Cardiovirus/tratamento farmacológico , Infecções por Cardiovirus/prevenção & controle , Infecções por Cardiovirus/virologia , Núcleo Celular/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Vírus da Encefalomiocardite , Endotoxemia/tratamento farmacológico , Endotoxemia/etiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-1/biossíntese , Interleucina-1/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Miocardite/tratamento farmacológico , Miocardite/virologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Piperidinas/uso terapêutico , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
It has been reported that co-delivery of IL-12 DNA with a DNA vaccine further enhances antigen (Ag)-specific protective immunity in pathogenic challenge models. However, the enhancing effects of antibody by IL-12 have been controversial. To clarify this issue, we constructed an IL-12 expression vector, co-immunized IL-12 DNA with an encephalomyocarditis virus (EMCV)-D VP1 plasmid vaccine, and then evaluated immune modulatory effects and protection against lethal EMCV-K challenge. We observed that VP1-specific IgG production, as well as seroconversion rates, were significantly enhanced by IL-12 co-injection, indicating that IL-12 can enhance antibody responses in this model system. In particular, co-injection with VP1 plus IL-12 DNA into the same leg enhanced systemic Ag-specific IgG production to a significantly greater extent than either the separate leg injection of VP1 and IL-12 DNA or VP1 DNA vaccine alone. This suggests that local co-expression of IL-12 along with antigens is more important for enhanced antibody production. Furthermore, IgG2a isotype was significantly enhanced by IL-12 DNA co-injection, indicating a Th1 bias. In addition, co-delivery of IL-12 DNA was demonstrated to enhance VP1-specific Th cell proliferative responses. When animals were challenged with a lethal dose of EMCV-K, IL-12 DNA-co-immunized animals exhibited enhanced survival, as compared to VP1 DNA vaccine alone. These studies suggest that IL-12 plays an important role in increasing Ag-specific Th1 type antibody and cellular responses, resulting in enhanced protection against lethal EMCV-K challenge.