Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice.
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Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM. (+info)
Viral myocarditis and dilated cardiomyopathy: mechanisms, manifestations, and management.
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Viral infection of the heart is relatively common and usually of little consequence. It can, however, lead to substantial cardiac damage and severe acute heart failure. It can also evolve into the progressive syndrome of chronic heart failure. Recent studies have gone some way towards unravelling the complex mechanisms underlying the heart muscle damage that occurs after viral infection. These studies have lent support to both immune and viral mediated (independent of an immune response) cardiac damage. Acute myocarditis can present in various ways, and it may be a cause of sudden death in an otherwise healthy young adult. New treatments for viral heart disease are awaited. In the meanwhile, the haemodynamic support of patients with acute left ventricular failure caused by myocarditis should be aggressive, to allow for the possibility of spontaneous recovery. Contemporary trials of treatment in chronic heart failure secondary to dilated cardiomyopathy support the use of angiotensin converting enzyme inhibitors, beta adrenoceptor blockers, and spironolactone in such patients. (+info)
Cardioselective infection with coxsackievirus B3 requires intact type I interferon signaling: implications for mortality and early viral replication.
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BACKGROUND: Interferons (IFNs) play an important role in antiviral defense and have therapeutic potential in coxsackievirus heart disease. However, little is known about the relative contributions of type I and type II IFN signaling in coxsackievirus B3 (CVB3) infection or their role in the cardioselective nature of CVB3 infection. METHODS AND RESULTS: Wild-type mice and mice deficient for either the type I or the type II IFN receptor (IFNR) were infected with CVB3. Infection of the type I IFNR-deficient mice with >10(3) plaque-forming units (pfu) of CVB3 resulted in 100% mortality within 2 to 4 days after infection. Death was rare in wild-type and type II IFNR-deficient mice after inoculation with as much as 10(8) pfu of CVB3. Surprisingly, the early mortality in the type I IFNR-deficient mice was not accompanied by higher virus titers in the heart. Unexpectedly, a dramatic increase of viral RNA in the liver was found to correlate with early mortality in type I IFNR-deficient mice. CONCLUSIONS: Type I but not type II IFN signaling is essential for the prevention of early death due to CVB3 infection. Interestingly, neither type I or type II IFN signaling has a dramatic effect on early viral replication in the heart. However, lethal viral replication in the liver is controlled by type I IFNs. These results demonstrate that the IFN system is capable of modulating both viral pathogenicity and tissue tropism. (+info)
Transcription factor activating transcription factor (ATF)-2 is activated by inflammatory signals transduced by the JNK and p38 MAP kinase pathways. To better define the role of ATF-2 in inflammation, adult mice expressing small amounts of a mutant ATF-2 protein were challenged with lipopolysaccharide (LPS), anti-CD3 antibody or virus. Within 3 h of challenge by LPS, ATF-2 mutant mice had decreased induction of the adhesion molecules E-selectin, P-selectin and VCAM-1 as well as the cytokines tumor necrosis factor-alpha, IL-1beta and IL-6 compared with control mice. Stimulation of T lymphocytes by anti-CD3 antibody also showed less induction of IL-1 and IL-6 in ATF-2 mutant tissues. ATF-2 mutant thymocytes treated with anti-CD3 antibody in vitro demonstrated reduced induction of c-Jun, JunB, JunD and Fra-2. However, similar to what was observed after p38 kinase inhibition in normal mice, relative ATF-2 deficiency did not prevent the development of a mononuclear cell infiltrate in the week following an inflammatory stimulus. ATF-2 mutant mice proved more susceptible to death than control mice from LPS plus D-galactosamine injection or Coxsackievirus B3 infection and had a higher incidence of mononuclear pulmonary infiltrates after exposure to Herpes simplex virus-1. ATF-2 is essential for maximal immediate induction of adhesion molecules and cytokine genes, but at later time points may even protect against overactive immune responses. (+info)
Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection.
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Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen. (+info)
A critical role for inducible nitric oxide synthase in host survival following coxsackievirus B4 infection.
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Coxsackieviral infections have been linked etiologically to multiple diseases. The serotype CB4 is associated with acute pancreatitis and autoimmune type 1 diabetes. To delineate the mechanisms of host survival after an acute infection with CB4 (strain E2), we have investigated the role of nitric oxide (NO), generated by the inducible form of nitric oxide synthase (NOS2), in viral clearance and pancreatic beta-cell maintenance. Mice deficient in NOS2 (NOS2-/- mice) and their wild-type (wt) counterparts were injected with CB4, after which both groups developed severe pancreatitis, hepatitis, and hypoglycemia within 3 days. Within 4 to 7 days postinfection (p.i.), most of the NOS2-/- mice died and at a strikingly higher mortality rate than wt mice. Histological examination of pancreata from both infected NOS2-/- and infected wt mice revealed early and complete destruction of the pancreatic acinar tissue, but intact, insulin-stained islets. When examined up to 8 weeks p.i., neither surviving NOS2-/-mice nor surviving wt mice developed hyperglycemia. However, the clearance of infectious CB4 was different between the mice. The spleens of NOS2-/- survivors were cleared of infectious virus with kinetics similar to that of wt mice, but the livers, pancreata, kidneys, and hearts of the NOS2-/- groups cleared virus more slowly than those of the wt group. This delayed clearance was particularly prominent in the livers of infected NOS2-/- mice, which also showed prolonged histopathological features of viral hepatitis. Taken together, this outcome suggests that NOS2 (and NO) is not required for the prevention of pancreatic beta-cell depletion after CB4 infection. Instead the critical actions of NOS2 apparently occur early in the host immune response, allowing mice to survive and clear virus. Moreover, the data support the existence of an organ-specific dependency on NO for a rapid clearance of CB4. (+info)
Altered expression of Bag-1 in Coxsackievirus B3 infected mouse heart.
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OBJECTIVE: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. METHODS: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. RESULTS: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. CONCLUSION: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease. (+info)
Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein.
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Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells. (+info)