Temperature-Sensitive Mutant of Coxsackievirus B3 Establishes Resistance in Neonatal Mice That Protects Them During Adolescence Against Coxsackievirus B3-Induced Myocarditis
Authors: Gauntt Charles J. , Paque Ronald E. , Trousdale Melvin D. , Gudvangen Richard J. , Barr David T. , Lipotich George J. , Nealon Timothy J. , Duffey Paul S. 
Published: January 2020

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.