The goals of this proposal are to identify mechanisms by which a small nonenveloped RNA genome virus induces severe focal inflammation in heart tissues of mice, as a model of human disease myocarditis. Most human patients with chronic myocarditis produce autoantibodies to antigens on cardiac tissue. In a murine model of coxsackievirus B3 (CVB3)-induced chronic myocarditis, autoantibodies are also produced against cardiac cell antigens. Administration of murine sera containing CVB3-neutralizing antibodies into CVB3-inoculated mice at 3 days post-inoculation exacerbates myocarditis, suggesting the presence of pathologic antibodies. Preliminary studies of neutralizing monoclonal antibodies (mAbs) against CVB3 demonstrate shared epitopes between CVB3 particles and cultured murine cardiac fibroblasts, as assessed by antibody binding studies, complement- mediated lysis of fibroblasts and stimulation of synthesis of a macrophage chemoattractant by fibroblasts. One mAb induces myocarditis in normal mice. Mechanisms by which eight mAbs could contribute to pathogenesis of myocarditis in mice will be identified. Induction of myocarditis by mAB will be confirmed. The capacity of mAbs for participating in complement- or killer cell-mediated lysis of murine cardiac fibroblasts will be quantitatively assessed. mAb-enhanced production of soluble factors from cardiac fibroblasts will be quantified. The viral polypeptide(s) to which the eight mAbs bind will be identified. Several laboratories have shown that enteroviral genomes are present in heart tissues of 15-25% of patients with myocarditis when infectious virus cannot be detected. CVB3 murine models of chronic myocarditis will be used to determine whether viral genomes persist in heart tissues, using in situ transcription and polymerase chain reaction. To understand the molecular basis of CVB3- induced myocarditis, the genomes of highly myocarditic (CVB3m) and amyocarditic (CVB3o) variants will be cloned. Reciprocal recombinant viruses will be generated by construction of hybrid genomes from infectious cDNA molecules of each genome and assessed for myocarditis-inducing ability in adolescent mice. Infectious hybrid cDNA molecules will be constructed with progressively smaller nucleotide regions associated with myocarditis to generate recombinant viruses. The nucleotide regions associated with myocarditis will be sequenced. CVB3 infection of some murine strains induces T cells which lyse uninfected target cells. Experiments will determine whether T lymphocytes from CVB3m-inoculated mice with chronic myocarditis will proliferate in vitro in response to soluble antigens extracted from normal murine hearts/cultured cardiac fibroblasts and whether extent of proliferation is predictive of severity of myocarditis.
