Temperature-Sensitive Mutant of Coxsackievirus B3 Establishes Resistance in Neonatal Mice That Protects Them During Adolescence Against Coxsackievirus B3-Induced Myocarditis

Abstract

Inoculation of neonatal CD-1 mice by multiple routes with an amyocarditic temperature-sensitive ( ts ) mutant ( ts 1) derived from a myocarditic parent variant of coxsackievirus B3 (CVB3 m ) resulted in approximately half of the neonates surviving to adolescence. Challenge of the ts 1 survivors with CVB3 m did not induce myocarditis, as assessed by histological examination of heart tissues. Virus was not detected in heart tissues of adolescent ts 1 survivors, but inoculation of these mice with CVB3 m resulted in virus concentrations similar in titers to those found in CVB3 m -inoculated normal adolescent mice. The ts 1 survivors did not contain detectable levels of anti-CVB3 m neutralizing antibody, but upon challenge with CVB3 m they produced antibody more rapidly and to higher titers than did normal CD-1 adolescents after primary inoculation with CVB3 m . Cell-mediated immunity in ts 1 survivors was compared with that of normal mice after challenge with CVB3 m . The capacity for production of migration inhibitory factor was assessed by the agarose droplet cell migration inhibition assay, using peritoneal exudate cells and a CVB3 m cell lysate or KCl-extracted antigens from heart tissues of CVB3 m -inoculated mice. Migration inhibitory factor activity was not detected in cultures of splenic leukocytes from ts 1 survivors of CVB3 m -inoculated ts 1 survivors, but it was readily detected in cultures of splenic leukocytes from CVB3 m -inoculated normal adolescent mice. The [ 3 H]thymidine stimulation assay, performed with splenic lymphoid cells and purified CVB3 m particles, revealed that lymphocytes from normal mice, whether inoculated with CVB3 m or not, were not stimulated by CVB3 m particle antigens, whereas lymphoid cells from a significantly higher proportion of ts 1 survivors, whether inoculated with CVB3 m or not, responded with a stimulation index ≥2.0. The cells responding with positive stimulation were T lymphocytes. A higher proportion of normal mice and ts 1 survivors, both inoculated with CVB3 m , contained splenic cytotoxic T lymphocytes with higher reactivity against CVB3 m -infected neonatal skin fibroblasts than against normal skin fibroblasts, as assessed by a 51 Cr release assay. The group of uninoculated ts 1 survivors present as a high proportion of individuals with cytotoxic T-lymphocyte reactivity against both uninoculated and CVB3 m -inoculated skin fibroblasts. However, ts 1 survivors and normal mice possessed the same proportions of splenic lymphocytes carrying either allele for Lyt 1 and Lyt 2 surface markers. The results suggest two mechanisms by which ts 1 survivors exhibit resistance to CVB3 m induction of myocarditis, namely, the rapid production of high-titered anti-CVB3 m neutralizing antibody in response to CVB3 m inoculation and altered cell-mediated immune responses against CVB3 m -induced viral or novel cellular antigens. The data are compatible with the notion that an immune deviation mechanism, thought to be controlled through a mechanism requiring suppressor cell activity which inhibits macrophage activation in ts 1 survivors, protects these mice from induction of myocarditis.