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.

Cell-associated lipoteichoic acids (LTAs) from late-exponential-phase cultures (serotypes Ia, Ib, Ic, II, and III) of group B streptococci isolated from infected and asymptomatically colonized infants were quantitated and characterized by growing the organisms in a chemically defined medium containing [3H]glycerol and [14Clacetate. Cell pellets were extracted with 45% aqueous phenol and chloroform-methanol and subjected to DEAE-Sephacel anion-exchange chromatography. Elution profiles resolved three major peaks, I, II, and III, with glycerol and phosphate present in a 1:1 molar ratio in each peak, and results obtained by Ouchterlony immunodiffusion analysis confirmed the presence of poly(glycerol phosphate). Saponification indicated that [14C]acetate was incorporated into fatty

Lipoteichoic acids (LTA) of serotype III strains of group B streptococci (GBS) were shown to mediate adherence of these organisms to human embryonic (HEC), fetal (HFC), and adult buccal (HBEC) epithelial cells. The binding of GBS was temperature dependent, and maximum attachment occurred at 37°C. HEC, HFC, and HBEC preincubated with purified LTA significantly inhibited attachment of GBS, whereas the group B and type III antigens had no effect. Under phosphate-limiting conditions in which cell-associated LTA could not be detected in these organisms, bacterial adherence did not take place. GBS (virulent) that were isolated from infected infants and previously shown to have significantly higher quantities of cellassociated LTA in comparison to GBS strains from asymptomatically colonized infants adhered with greater binding avidity to HEC and HFC and in greater numbers than to HBEC. It was determined that the mechanism of LTA-mediated adherence of GBS to HBEC differed from adherence to embryonic and fetal cells for both virulent and asymptomatic GBS strains bound to HBEC in a similar manner, enhanced by the lipid portion of the LTA. In contrast, the binding of GBS to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate polymer of LTA. These results indicate that possible receptor sites for LTA present on cells in prenatal stages of development may differ from those of adult cells, which may result in increased susceptibility of newborn infants to group B streptococcal disease. The implications of LTA-mediated adherence of GBS and their possible role as virulence factors are discussed.

The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype Ill strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA

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.