José R. Romero

Mark A. Pallansch

Nora M. Chapman

Steven Tracy

English

Crossref

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Public Health Resources Public Health Resources 
1997 
Sites Other Than Nucleotide 234 Determine Cardiovirulence in 
Natural Isolates of Coxsackievirus B3 
Nora M. Chapman 
University of Nebraska Medical Center, Omaha, Nebraska 
Jose R. Romero 
University of Nebraska Medical Center, Omaha, Nebraska 
Mark A. Pallansch 
Centers for Disease Control and Prevention, Atlanta, Georgia 
Steven Tracy 
University of Nebraska Medical Center, Omaha, Nebraska 
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Chapman, Nora M.; Romero, Jose R.; Pallansch, Mark A.; and Tracy, Steven, "Sites Other Than Nucleotide 
234 Determine Cardiovirulence in Natural Isolates of Coxsackievirus B3" (1997). Public Health Resources. 
7. 
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Sites Other Than Nucleotide 234 Determine
Cardiovirulence in Natural Isolates of
Coxsackievirus B3
Nora M. Chapman,1* José R. Romero,2 Mark A. Pallansch,3 and Steven Tracy1
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
2Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska
3Enterovirus Diagnostic Laboratory, Division of Viral and Rickettsial Diseases, Centers for Disease Control and
Prevention, Atlanta, Georgia
The genetic site(s) that naturally determine the
cardiovirulence phenotype of coxsackievirus B3
(CVB3) have yet to be mapped. Using two
closely related CVB3 strains that differed in
terms of cardiovirulence phenotype in mice, we
previously reported the difference in phenotype
mapped to a single site, nucleotide 234 (nt234) in
the 58 non-translated region (NTR) of the CVB3
genome. When nt234 was C, the virus was at-
tenuated and when U, the virus was cardioviru-
lent. To determine whether this finding was ap-
plicable to other strains of CVB3, we examined
13 different naturally occurring CVB3 strains iso-
lated in different years in the United States. We
determined that only two isolates induced se-
vere inflammatory heart muscle disease in C3H/
HeJ male mice. Using PCR products as sequenc-
ing templates, we determined the 58 NTR se-
quence from each viral genome. Alignment of
these sequences and other published CVB3 58
NTR sequences suggests as many as four sepa-
rate lineages, with commonly used laboratory
strains clustering closely in one branch. An ex-
amination of the sequences showed that regard-
less of cardiovirulence phenotype, nt234 was in-
variably uridine. Thus, the previously reported
cytidine at nt234 is most likely the result of a rare
mutation and is not a naturally occurring varia-
tion and other sites must account for the vari-
ance in virulence seen in natural isolates of
CVB3. J. Med. Virol. 52:258–261, 1997.
© 1997 Wiley-Liss, Inc.
KEY WORDS: coxsackievirus B3; virulence;
58 non-translated region
INTRODUCTION
Coxsackievirus B3 (CVB3) is a typical human entero-
virus (Picornaviridae) with a 7,400 nucleotide genome
including a 741 nucleotide 58 non-translated region
(NTR) [Tracy et al., 1996; Klump, 1990; Tracy et al.,
1992; Chapman et al., 1994]. Coxsackievirus B3 is a
cause of inflammatory heart muscle disease in both
children and adults on the basis of isolation of infec-
tious virus and serological studies [reviewed in Mar-
tino et al., 1995]. In addition, CVB3-induced murine
acute inflammatory heart muscle disease (myocarditis)
resembles many aspects of the homologous disease in
man [Woodruff, 1980; Tracy et al., 1996].
Using two genetically similar strains of CVB3 that
differed by murine cardiovirulence phenotype, we pre-
viously identified nucleotide 234 (nt234) in the 58 NTR
of the CVB3 genome as the primary determinant of the
phenotype for these two viral strains [Tu et al., 1995].
The avirulent strain, CVB3/0 [Gauntt et al., 1979], is
genotypically differentiable from the cardiovirulent
strain, CVB3/20 [Tracy et al., 1992], in having a C
rather than a U at nt234 [Chapman, 1994], a change
that results in an attenuated phenotype. To determine
the applicability of these findings to other CVB3
strains, we examined naturally occurring strains of
CVB3 for cardiovirulence phenotype in mice and the
identity of nt234.
MATERIALS AND METHODS
Virus Strains
Thirteen natural isolates of CVB3 were used for this
study. Virus isolates were derived from different years
in the United States. Strains AS, CO, DO, GA, GU, OL,
RE, ZU were the kind gift from D. Schnurr, California
Department of Health Sciences, Berkeley, CA [Gauntt
and Pallansch, 1996]. Strains 1699, 5965, D7783, 3175,
8680 were obtained from the Centers for Disease Con-
trol and Prevention [Gauntt and Pallansch, 1996]. All
*Correspondence to: Nora M. Chapman, Department of Pathol-
ogy and Microbiology, University of Nebraska Medical Center,
Omaha, NE 68198.
Accepted 29 January 1997
Journal of Medical Virology 52:258–261 (1997)
© 1997 WILEY-LISS, INC.
stocks were prepared from low MOI infections, titered
and stored frozen in aliquots until use.
Inoculation of Mice
C3H/HeJ male mice (Jackson Laboratories) at 3–4
weeks of age were rested until body weights were be-
tween 12–15 g per mouse. Groups of five mice were
inoculated intraperitoneally with 2 × 105 TCID50 virus
in 0.1 ml unsupplemented medium, then sacrificed 10
days later. Mouse hearts were formalin fixed, then em-
bedded in paraffin. Six 5–6-m thick sections were cut,
with each section separated by five to six similarly cut
sections. Hematoxylin and eosin stained heart sections
were examined by light microscopy for the presence of
inflammatory lesions and muscle damage.
Nucleotide Sequence Analysis
Virion RNA was prepared for sequence analysis us-
ing IsoQuick (ORCA Research, Inc.). Following resus-
pension of viral RNA in reverse transcription mix,
cDNA was generated as described earlier using primer
JRpATG [Romero and Rotbart, 1995; 58-ACTTGAG-
CTCCCATT, reverse complement of CVB3/20 nucleo-
tides (nt) 742–756, numbering the 58 terminal nucleo-
tide as 1; [Chapman et al., 1994]. Ten microliters of a
reverse transcriptase reaction was used directly in a
PCR as described [Romero and Rotbart, 1995] using
the primers JRp65 (58-ACGGTACCTTTGTGCGCCT-
GTTTT; CVB3/20 nt 63–86) and JRpATG to generate a
694 bp fragment comprising the 58NTR from nt63
through 756. Bands of expected size were excised from
agarose gels, purified using GeneClean (Bio101, Inc.)
and used as template for sequence analysis. Sequenc-
ing was accomplished using the Cyclist Exo-Pfu DNA
Sequencing Kit (Stratagene). Sequencing gels were
dried and exposed to Kodak X-Omat film. Sequences
were analyzed using the GCG suite of software (Genet-
ics Computer Group, Inc., Madison, WI). The 58 NTR
sequences derived for this study have been posted on
and may be downloaded from the worldwide web at
http://www.unmc.edu/Pathology/Myocarditis.
RESULTS
Of thirteen naturally occurring strains of CVB3, two
were cardiovirulent (strains AS and DO). These viruses
induced large, widespread inflammatory lesions con-
taining necrotic regions and calcification in every
mouse heart (Fig. 1a). For the remainder of the virus
strains examined, in general no lesions were observed
in any of the six sections examined. A few viruses in-
duced a few, small (about five to 20 infiltrating cells)
lesions with no necrosis or calcification in a minority of
the hearts (Fig. 1b,c).
The 58 NTR sequence from nt 87–741 [relative to
CVB3-20; Tracy et al., 1992] from each viral strain was
determined. The relationships of these sequences with
those of other published CVB3 58NTR sequences were
also compared (Fig. 2) using alignments and a dendro-
gram generated using the PILEUP program (Genetics
Computer Group, Inc.) which uses progressive pairwise
alignments between sequences to generate a final
alignment. Four potential lineages (A, B, C, D) can be
distinguished with sequence identities greater than
87% within the group. The C lineage contains the five
primary laboratory strains, in addition to the AS
strain. Five of the six strains in this cluster are cardio-
virulent and when nt234C in the only avirulent strain
of this group, CVB3/0, reverts to U, this strain is viru-
lent as well [Tu et al., 1995; Tracy et al., 1996]. Selec-
tion of cardiovirulent strains in murine models of myo-
carditis from strains isolated in one period of time may
Fig. 1. Typical examples of thin sections of murine hearts from
C3H/HeJ male mice infected with (a) cardiovirulent strain CVB3/AS
or (b) avirulent strain CVB3/ZU at day 10. Arrowhead denotes small
lesion in (b), which is seen at higher magnification (c). Hematoxylin
and eosin stained, 100× (a,b) or 400× (c).
Cardiovirulence in Natural Isolates of Coxsackievirus B3 259
have been responsible for this clustering of genetically
similar cardiovirulent strains.
Despite the sequence differences in this region of
each CVB3 isolate and regardless of the cardioviru-
lence phenotype, examination of these CVB3 sequences
revealed that nt234 was uridine in each sequence. Fur-
thermore, the 5 nucleotide sequence encompassing
nt234 described by CVB3 nt232–236 (58-CGUUA) was
invariable not only among CVB3 but all other human
enteroviral sequences examined as well (Genbank Re-
lease 96.0, 8/96). When this 5mer was located on pub-
lished global secondary structures of the 58 NTRs of
enteroviruses [Skinner et al., 1989; Le and Zuker,
1990], it was found to be between the primary domains
III and IV [Minor, 1992].
DISCUSSION
Assay of cardiovirulence of the wild-type CVB3
strains in C3H/HeJ mice revealed that two of 13 CVB3
strains were significantly cardiovirulent in all mice
tested, while the remaining 11 strains were minimally,
if at all, cardiovirulent. Two previous studies examined
many of these same strains in the CD-1 mouse strain
[Tracy and Gauntt, 1987; Gauntt and Pallansch, 1996].
These studies suggest that the great majority of CVB3
strains examined are non-cardiovirulent when tested
in mice. The strain CVB3/AS (Fig. 1a) produced myo-
carditic lesions in both CD-1 and C3H mice, while the
strain CVB3/ZU was scored as virulent in studies in
CD-1 mice [Tracy and Gauntt, 1987; Gauntt and Pal-
lansch, 1996] but produced 10-fold fewer lesions than
AS and was one of the strains in the present study that
was avirulent (Fig. 1b,c). Although the mouse strain
used can greatly affect the degree of disease induced by
cardiovirulent CVB3 [Khatib et al., 1987], C3H/HeJ
male mice are highly sensitive to CVB3-induced myo-
carditis [Kiel et al., 1989; Tracy et al., 1992]. Greater
susceptibility to CVB3-induced myocarditis of CD-1
mice may account for the cardiovirulence difference of
CVB3/ZU in this study and previous studies.
Sequence analysis of these CVB3 strains demon-
strated that nt234 was invariably uridine. Due to this
invariance of sequence in CVB3 strains regardless of
the established pathogenicity of the virus, nt234U can-
not be a determinant of naturally occurring cardiovi-
rulence for CVB3 and must be viewed as a mutation.
The 5mer sequence encompassing nt234 (CVB3 context
nt232–234, 58-CGUUA) is completely conserved in all
published human enterovirus sequences and in these
CVB3 strains. This 5mer is not located within an es-
tablished secondary structure domain [Skinner et al.,
1989; Le and Zuker, 1990], but is included within the
boundaries of the internal ribosome entry site critical
for enterovirus translation as defined in studies of po-
lioviruses [Meerovitch et al., 1991; Nicholson et al.,
1991; Pestova et al., 1991] and the site of major deter-
minants of poliovirus attenuation [reviewed in Minor,
1992]. Despite this location, analysis of CVB3/0 and
CVB3/20 infected murine fetal heart fibroblasts shows
no significant differences in viral protein translation
[Tu et al., 1995; Tracy and Chapman, unpublished ob-
servations]. However, viral transcription in murine
and human cardiac cell cultures is significantly dis-
turbed when nt234 is present as C [Tu et al., 1995] or
G [S. Tracy, N. Chapman, J. Romero, unpublished
data]. This site, and perhaps other sites within the
5mer, may represent a useful site(s) for targeting mu-
tations to artificially attenuate CVB3 and possibly
other enteroviruses. That natural CVB3 isolates vary
in cardiovirulence and yet are completely conserved in
sequence in this region indicates sites elsewhere in the
genome are important for determination of cardioviru-
lence in CVB3 strains circulating in the human popu-
lation.
ACKNOWLEDGMENTS
We thank J. Smith Leser and Melanie Fron for ex-
pert technical assistance. This work was supported by
grants from the Public Health Service (N.M.C., J.R.R.,
S.T.), the American Heart Association (N.M.C., S.T.),
the Edna Ittner Foundation (J.R.R.).
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Cardiovirulence in Natural Isolates of Coxsackievirus B3 261


Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

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Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

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