Inoculation of adolescent CD-1 mice with one variant of coxsackievirus B3 (CVB3m) results in induction of readily observable myocardial lesions, whereas inoculation of siblings with a second variant (CVB3o) results in little or no myocarditis. These variants could not be distinquished from each other on the basis of replication properties in HeLa cells or cardiac tissues in vivo, sensitivity to human interferon in HeLa cells, induction of interferon in the mouse, generation of detectable levels of defective-interfering particles in HeLa cells or in cardiac tissue in vivo, stimulation of serum-neutralizing antibody titers, nor in their rate of clearance by the spleen. Infectivity of CVB3o was slightly more heat labile at 34 degrees C than CVB3m. Little if any replication of either CVB3o or CVB3m occurred in either adherent or nonadherent populations of normal murine lymphoid cells. Cardiac tissues from mice inoculated with CVB3m but not CVBo contain new antigens that can inhibit migration of sensitized lymphocytes from CVB3m-immunized mice in an in vitro cell-migration-inhibition assay. However, the CVB3o variant was shown to have the genetic capability of inducing myocarditis if the mice were treated with cyclophosphamide prior to virus inoculation. These results suggest, in agreement with our previously published work, that induction of myocarditis by CVB3 requires destruction of myocytes by virus and subsequent stimulation of cell-mediated responses to new antigens produced in the myocardium during virus replication.

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Pre-treatment of syngeneic mice with polyclonal populations of Protein-A separated anti-idiotypic antibodies prepared against anti-CVB3 viral idiotypes resulted in reduction of inflammatory myocarditis in virus-challenged mice. Cellular immunity as assessed by cell-migration-inhibition resulted in specific cell-mediated sensitivity against anti-Ids (CVB3) as well as viral preparations. Animals pre-treated with anti-idiotypic preparations developed an anamnestic anti-viral antibody response, with antibodies capable of specifically binding CVB3 virus antigen in an ELISA assay; but without CVB3 viral neutralizing capability. Adoptive transfer of limited numbers of syngeneic anti-Id immunized lymphoid cell populations failed to alter the course of inflammatory myocarditis in CVB3 virus-challenged animals. Cellular binding studies utilizing anti-Ids suggested increased, but nonspecific binding of anti-Ids to lymphoid and myocyte populations in CVB3 infected animals. The data suggest an immunomodulatory role of idiotype-anti-idiotype interactions in the development of myocarditis.

Ten temperature-sensitive ( ts ) mutants isolated from a myocarditis-inducing wild-type (WT) coxsackievirus B3 parent did not induce myocarditis in adolescent CD-1 mice. An avirulent prototype ts mutant from one of the three complementation groups adsorbed to murine cardiac tissue, as did WT virus. Heart tissues from mice inoculated with WT virus contained 100- to 1,000-fold more virus than heart tissues from mice inoculated with any of the three prototype ts mutants. WT virus exhibited a greater capsid stability and a higher efficiency of replication at 37°C than any of the three prototype ts mutants. All three prototype ts mutants induced less interferon in vivo than WT virus. Cell-mediated immune responses, assessed by the cell migration inhibition assay, were different in mice inoculated with WT virus when compared to ts 5 mutant virus. Peritoneal exudate cells from mice inoculated with WT but not ts 5 virus reacted specifically against antigens in WT virus HeLa cell lysates and antigens extracted with KCl from cardiac tissues of mice inoculated with WT virus. Cardiac tissues of mice inoculated with WT but not ts 5 virus contained KCl-extractable antigens which were able to specifically inhibit the migration of peritoneal exudate cells taken from mice immunized with WT virus. Therefore, ts 5 neither elicited a measurable cell-mediated immune response nor induced antigens in cardiac tissues which were immunoreactive with sensitized-(WT virus)-peritoneal exudate cells. Of 9 revertant viruses isolated from the 10 ts mutants, 5 showed covariance in ability to replicate at 39.5°C and capacity for induction of myocarditis. Some revertants exhibited a reduced capsid thermostability compared to WT virus but yet retained the capacity for induction of myocarditis. The data suggest that induction of myocarditis by coxsackievirus B3 variants depends on a combination of several variables, including capsid stability, capacity for replication at 37°C, and expression of the three identified genes. All three prototype ts mutants served as vaccine viruses in preventing myocarditis in adolescent mice subsequently challenged with WT virus. However, all three prototype ts mutants and their revertant variants retained partial to complete lethality in CD-1 neonates.

Investigation into the mechanism(s) whereby monocytes-macrophages are mobilized to the myocardium of coxsackievirus (CVB3)-infected mice was approached by assessing the role of anti-idiotypic antibodies against specific antiviral IgG molecules. Balb/c mice preinoculated with polyclonal anti-idiotypic, syngeneic antibodies resulted in significantly decreased myocarditis after virus challenge. On the other hand, oil-induced peritoneal exudate cells obtained from virus-exposed animals demonstrated increased chemotaxis in the presence of polyclonal anti-Id preparations, whereas peritoneal exudate cells from normal animals did not. Specificity of the anti-Ids as confirmed by the binding of the syngeneic anti-anti-idiotypic antibodies (antibody 3) to solubilized viral antigen(s) and Fab fragments prepared from the anti-idiotypic antibodies. Paradoxic biological effects of anti-Id, demonstrated in vitro and in vivo, suggest possible and subtle immunoregulation of the macrophage effector arm by anti-idiotypes during the course of virus infection.