Viruses Cause Type 1 Diabetes

in Animals
JI-WON YOON AND HEE-SOOK JUN
Rosalind Franklin Comprehensive Diabetes Center, Chicago Medical School,
North Chicago, Illinois 60064, USA

ABSTRACT: More than 10 viruses have been reported to be associated

with the development of type 1 diabetes-like symptoms in animals, with
the best evidence coming from studies on the D variant of encephalomyocarditis (EMC-D) virus in mice and Kilham rat virus (KRV) in rats. A
high titer of EMC-D viral infection results in the development of diabetes
within 3 days, primarily due to the rapid destruction of b cells by viral
replication within the cells. A low titer of EMC-D viral infection results
in the recruitment of macrophages to the islets. Soluble mediators produced by activated macrophages play a critical role in the destruction of
residual b cells. A single amino acid at position 776 of the EMC viral
genome controls the diabetogenicity of the virus. In contrast, KRV causes
autoimmune type 1 diabetes in diabetes-resistant BioBreeding (DR-BB)
rats without direct infection of b cells. Macrophages play an important
role in the development of diabetes in KRV-infected DR-BB rats. As well,
KRV infection preferentially activates effector T cells, such as Th1-like
CD45RC+ CD4+ T cells and CD8+ T cells, and downregulates regulatory T cells, such as Th2-like CD45RC CD4+ T cells. This results in the
breakdown of the immune balance, contributing to the development of
diabetes in KRV-infected DR-BB rats.
KEYWORDS: type 1 diabetes; Kilham rat virus; encephalomyocarditis
virus; macrophages; T cells; cytokines; pancreatic b cells

INTRODUCTION
Over the last several decades, at least 10 viruses have been reported to be
associated with the development of type 1 diabetes-like syndromes in animals.
These viruses are coxsackie B viruses in mice and/or nonhuman primates,
encephalomyocarditis (EMC) virus in mice, mengo virus in mice, foot-andmouth disease virus in pigs and/or cattle, retrovirus in mice, rubella virus in
hamsters and rabbits, bovine viral diarrhea-mucosal disease virus in cattle,
reovirus in mice, Kilham rat virus (KRV) in rats, and cytomegalovirus in the
Address for correspondence: Hee-Sook Jun, Rosalind Franklin Comprehensive Diabetes Center,
Chicago Medical School, North Chicago, IL 60064. Voice: 847-578-8341; fax: 847-578-3432.
e-mail: hee-sook.jeon@rosalindfranklin.edu
C 2006 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1079: 138146 (2006). 
doi: 10.1196/annals.1375.021

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TABLE 1. Viruses associated with the development of type 1 diabetes in animals

Virus type
RNA viruses

DNA viruses

Virus
Coxsackie B virus
Encephalomyocarditis virus
Mengo virus
Foot-and-mouth disease virus
Retrovirus
Rubella virus
Bovine viral diarrhea-mucosal
disease virus
Reovirus
Kilham rat virus
Cytomegalovirus

Host

Involvement of
genetic factors

Mice, nonhuman
primates
Mice
Mice
Pigs, cattle
Mice
Hamsters, rabbits
Cattle

Yes
Yes
Yes
Not determined
Yes
Not determined
Not determined

Mice
Rats
Degu

Yes
Yes
Not determined

Degu (TABLE 1).1 Among those viruses, the most clear and unequivocal evidence that a virus induces type 1 diabetes in animals comes from studies on
EMC virus in mice2 and KRV in rats.3 EMC virus is considered to be a primary agent that is selectively injurious to pancreatic b cells, whereas KRV is
considered to be a triggering agent of b cell-specific autoimmunity without
infection of b cells. In this brief article, we will focus on these two models of
virus-induced diabetes.
EMC Virus-Induced Diabetes in Mice
EMC virus is a picornavirus with naked, single-stranded RNA and a genome
size of about 7.8 kB. The capsid comprises four polypeptides; VP1, VP2, VP3,
and VP4. EMC virus selectively infects pancreatic b cells and has been the
most thoroughly studied diabetogenic virus in animals. When genetically susceptible animals were infected with the M variant of EMC virus, diabetes was
inconsistently induced.4,5 Plaque purification of the M variant found two stable
variants, EMC-D and EMC-B, that could not be antigenically distinguished
by plaque neutralization assay, competitive radioimmunoassay, or molecular
hybridization studies with radiolabeled DNA complementary to EMC-D and
EMC-B RNAs. Nevertheless, the EMC-D virus produced type 1 diabetes in
over 90% of infected animals, whereas mice inoculated with EMC-B virus did
not acquire diabetes.6,7
Nucleotide sequence analysis showed that EMC-D virus (7829 bases) differs
from EMC-B virus (7825 bases) by only 14 nucleotides: two deletions totaling
5 nucleotides, 1 base insertion, and 8 point mutations.8 The first deletion
of 3 nucleotides and the second deletion of 2 nucleotides are located in the
5 -poly(C) tract and the 3 -end polyadenylation site, respectively. One base

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insertion in EMC-B occurs in the 5 -noncoding region. The 8 point mutations

are located in the polyprotein coding region. Two of these are silent, whereas
the remaining 6 mutations, 1 located on the L gene and 5 on the VP1 gene,
result in amino acid changes. Further studies of 21 different nondiabetogenic
and 15 different diabetogenic viruses derived from stocks of the EMC-B and
EMC-D variants revealed that only the 776th amino acid, alanine (Ala-776),
of the EMC virus polyprotein, located on the major capsid protein VP1, is
common to all diabetogenic variants. In contrast, threonine in this position
(Thr-776) is common to all nondiabetogenic variants.9
Point mutation at nucleotide position 3155 to substitute Ala-776 (GCC) with
various other amino acids, including threonine (ACC), serine (TCC), proline
(CCC), aspartic acid (GAC), or valine (GTC), resulted in the loss of diabetogenicity.10 Three-dimensional molecular modeling of the VP1 protein showed
greater van der Waals interactions and closer packing of residues surrounding
position 776 of the EMC virus polyprotein in viruses that contained Thr-776
than viruses that contained Ala-776. This results in more accessible surface areas surrounding Ala-776, which increases the number of binding sites for viral
attachment to b cell receptors and promotes viral infection, the destruction of
b cells, and subsequently the development of type 1 diabetes.11
The genetic background of the host also influences the diabetogenicity of
EMC-D virus, as only certain inbred strains of mice develop diabetes when
infected. Susceptibility to EMC-D virus appears to be determined by a single
autosomal recessive gene,12 which may operate by modulating the levels of
the expression of viral receptors on b cells.
Infection of genetically susceptible mouse strains with a high dose (5 105
plaque-forming units/mouse) of EMC-D virus results in the development of
diabetes within 34 days. Several lines of evidence show that the development
of diabetes in high-dose EMC-D virus-infected mice is mainly due to the
acute destruction of b cells by viral replication within the cells,5 rather than to
involvement of the immune system. Treatment of EMC-D virus-infected mice
with anti-L3T4 (anti-CD4) antibody and/or anti-Lyt2 (anti-CD8) antibody did
not alter the incidence of diabetes.13 In addition, athymic nude mice, which
lack functional lymphocytes, had an incidence of diabetes similar to their
heterozygous littermates when infected with EMC-D virus, indicating that
T cells do not play a significant role in the destruction of b cells by a high
dose of EMC-D virus.
Natural viral infections in animals and man generally involve exposure to
relatively low numbers of virus, rather than the high viral titers used in the
experiments described above. Thus, studies were conducted in mice infected
with a low dose (50100 plaque-forming units/mouse) of EMC-D virus.14
In contrast to the situation in high-dose-infected mice, it was found that the
immune system, especially macrophages, plays a central role in the destruction of pancreatic b cells in mice infected with a low dose of EMC-D virus.
Activation of macrophages prior to viral infection resulted in a significant

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increase in the incidence of diabetes, whereas inactivation of macrophages

prior to viral infection completely prevented EMC-D virus-induced diabetes.14
Further studies showed that exposure to a low dose of EMC-D virus resulted in initial, selective infection of pancreatic b cells and recruitment of
macrophages into the islets, followed by infiltration of other immunocytes including T cells, natural killer cells, and B cells.15 In this situation, replication
of the virus within the b cells plays a minor role in b cell destruction, whereas
the cascade of events initiated by macrophages causes most of the b cell
damage.16
To elucidate the mechanisms by which macrophages trigger b cell destruction in mice infected with a low dose of EMC-D virus, several studies examined the role of macrophage-derived soluble mediators, such as interleukin-1b
(IL-1b), tumor necrosis factor-a (TNF-a), and nitric oxide (NO). The expression of IL-1b, TNF-a, and inducible nitric oxide synthase (iNOS) was
detected in the pancreatic islets of mice infected with a low dose of EMC-D
virus, and inhibition of these molecules resulted in a significant decrease in the
incidence of diabetes.17 However, the molecular mechanism involved in the
destruction of b cells by these soluble mediators is not known. IL-1b induces
apoptosis through the induction of iNOS expression and NO production in rat
islet cells.18 Therefore, IL-1b and TNF-a, produced by activated macrophages
in the pancreatic islets after the infection of mice with a low dose of EMC-D
virus, may induce iNOS expression and NO production and contribute to b cell
death through apoptosis. Infection of macrophages isolated from DBA/2 mice
with EMC-D virus induced a significant expression of iNOS and a high level
of NO production.19 Further experiments showed that a tyrosine kinase signaling pathway is clearly involved in the EMC-D virus-induced activation
of macrophages, since inactivation of tyrosine kinase activity by treatment
of EMC-D virus-infected mice with a tyrosine kinase inhibitor, tyrphostin
AG126, prevented macrophage activation, resulting in the protection from
b cell destruction.19 Studies that examined the role of various tyrosine kinase families in EMC-D virus-induced macrophage activation revealed that
the Src family of kinases, p59/p56hck , plays a major role in the activation of
macrophages, which subsequently produce IL-1b, TNF-a, and NO, leading to
the destruction of pancreatic b cells.20
On the basis of these observations, it appears that infection of genetically
susceptible strains of mice with EMC-D virus initially results in the replication of the virus in pancreatic b cells and recruitment of macrophages that
are activated by the EMC-D virus into the pancreatic islets. These activated
macrophages produce soluble mediators, such as IL-1b, TNF-a, and NO in
the islets, which induce apoptosis in the b cells. The tyrosine kinase signaling
pathway, particularly the Src family of kinases, p59/p56hck , play a major role
in the activation of macrophages, resulting in the production of IL-1b, TNF-a,
and NO, leading to the destruction of b cells and subsequent development of
diabetes in mice.

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KRV-Induced Diabetes in Rats

KRV is a small DNA parvovirus that causes diabetes by inducing autoimmune responses against the b cell, rather than by direct b cell cytolysis, in
diabetes-resistant BioBreeding (DR-BB) rats. These rats are derived from
diabetes-prone progenitors, but do not normally develop the disease. When
infected with KRV at 3 weeks of age, about 30% of DR-BB rats develop autoimmune diabetes within 24 weeks and a further 30% show insulitis without
diabetes.3 However, the incidence of diabetes can be increased to 80100% if
DR-BB rats are injected with poly (I:C) along with KRV.3 It was later found
that activation of the innate immune system through toll-like receptor (TLR)
plays a crucial role in KRV-induced diabetes.21
KRV infects lymphoid organs, such as the spleen, thymus, and lymph nodes,
but not b cells. The mechanisms by which KRV causes the destruction of b cells
in DR-BB rats without infection are unclear. Macrophages appear to play an
important role in KRV-induced diabetes in DR-BB rats. The production of
macrophage-derived proinflammatory cytokines, such as TNF-a, IL-1b, and
IL-12 was closely correlated with an elevated Th1 immune response in KRVinfected DR-BB rats.22 Further studies found that NO produced from activated
macrophages played a critical role in the regulation of immune responses,
such as the upregulation of the Th1 immune response and activation of b cellspecific cytotoxic T lymphocytes, resulting in the development of autoimmune
diabetes in KRV-infected DR-BB rats.23
The presence of a common epitope between a KRV-specific peptide and
a b cell autoantigen, so-called molecular mimicry, has been suggested as
a mechanism for the initiation of b cell-specific autoimmune diabetes.3,22 If
this is the case, then KRV antigen-specific T cells generated by KRV peptides
might cross-react with b cells and attack them, resulting in the development of
insulitis and, subsequently, diabetes. However, experimental data showed that
infection of DR-BB rats with recombinant vaccinia viruses expressing the KRV
capsid proteins or nonstructural proteins did not cause insulitis or diabetes, even
though each viral peptide was clearly expressed in the infected DR-BB rats,
viral peptide-specific T cells were generated, and antibodies against the KRV
peptides were induced.24 This result suggests that molecular mimicry between
KRV peptides and b cell-specific autoantigens in DR-BB rats is unlikely to be
a mechanism by which KRV induces b cell-specific autoimmune diabetes.
An alternative hypothesis is that KRV infection disturbs the finely tuned
immune balance and activates autoreactive T cells that are cytotoxic to b
cells, resulting in T cells-mediated autoimmune diabetes similar to that seen in
diabetes-prone BioBreeding (DP-BB) rats. Several observations support this
hypothesis. In DR-BB rats infected with KRV, the absolute number of both
CD4+ and CD8+ T cells in splenocytes increased, however, the proportion of
CD4+ T cells in splenocytes decreased, whereas the proportion of CD8+ T cells
increased as a result of preferential proliferation of CD8+ T cells. In addition,

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treatment of KRV-infected DR-BB rats with OX-8 (anti-CD8) monoclonal

antibody decreased the incidence of diabetes.22 These results indicate that
CD8+ T cells are clearly involved in the destruction of b cells.
In the rat, CD4+ T cells can be divided into Th1-like CD45RC+ CD4+ T
cells, which express IL-2 and interferon-g (IFN-g) and play an important role
in cell-mediated immune responses, and Th2-like CD45RC CD4+ T cells,
which express IL-4 and IL-10 and play an important part in humoral immune
responses.25 It has been suggested that dominance of Th1 cells over Th2 cells
is associated with the development of autoimmune type 1 diabetes, whereas
the dominance of Th2 cells over Th1 cells is associated with the prevention
of type 1 diabetes.26 KRV infection in DR-BB rats significantly decreased the
number of Th2-like CD45RC CD4+ T cells and increased the number of Th1like CD45RC+ CD4+ T cells as compared with uninfected controls.24 Furthermore, adoptive transfer of both Th1-like CD45RC+ CD4+ T cells and CD8+
T cells, isolated from DR-BB rats after KRV infection, into young DP-BB
rats resulted in the development of diabetes in 88% of the recipients. The incidence of diabetes in DP-BB rats that received either CD45RC+ CD4+ or CD8+
T cells alone was significantly less than rats that received a combination of
CD45RC+ CD4+ and CD8+ T cells. A combination of CD45RC+ CD4+ T cells
from infected rats and CD8+ T cells from uninfected rats or a combination of
CD8+ T cells from infected rats and CD45RC+ CD4+ T cells from uninfected
rats did not change the incidence of diabetes.24 These results indicate that Th1like CD4+ and CD8+ T cells from KRV-infected rats work synergistically to
destroy b cells. In contrast, none of the recipients of both CD45RC CD4+ and
CD8+ T cells developed diabetes, indicating that CD45RC CD4+ T cells may
play a role as regulatory T cells. Additional studies also showed that the development of KRV-induced diabetes could be due to the failure to maintain the
function of regulatory T cells (CD4+ CD25+ T cells).27 On the basis of these
observations, it appears that infection of DR-BB rats with KRV results in the
preferential activation of effector T cells, such as Th1-like CD45RC+ CD4+
T cells and CD8+ T cells, and the downregulation of Th2-like CD45RC CD4+
and CD4+ CD25+ T cells, and the activated effector T cells kill the b cells,
similar to the case in DP-BB rats.
In this brief review of virus-induced diabetes, we have discussed how two
distinct viruses, a small RNA virus (EMC-D virus) and a small DNA parvovirus (KRV), can kill pancreatic b cells in mice and rats, resulting in type 1
diabetes. Infection of genetically susceptible strains of mice (SJL/J, SWR,
DBA/2) with a high dose of EMC-D virus results in the development of diabetes by the acute destruction of b cells through rapid replication of the virus
within the b cells. However, infection of genetically susceptible mouse strains
with a low dose of EMC-D virus results in the initial replication of the virus
in b cells, followed by recruitment of macrophages to the infected pancreatic
islets. These activated macrophages secrete soluble mediators, such as cytokines (IL-1, TNF-a, IFN-g) and oxygen-free radicals (NO), which destroy

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Figure 1. Two distinct pathogenic mechanisms of virus-induced type 1 diabetes in animals. KRV, a parvovirus, causes autoimmune diabetes without infection of b cells, whereas
EMC virus, a picornavirus, directly infects and destroys b cells. Infection of DR-BB rats
with KRV results in the activation of macrophages and breakdown of the finely tuned immune balance between Th1-like CD45RC+ CD4+ and Th2-like CD45RC CD4+ T cells,
resulting in the activation of b cell-specific, Th1-like CD4+ and CD8+ T cells. These cells
act synergistically to destroy b cells, which contributes to the development of diabetes. Infection of genetically susceptible strains of mice with a high dose of EMC-D virus destroys
b cells by direct cytolysis, and activated macrophages play a minor role in b cell destruction. However, infection with a low dose of EMC-D virus results in the initial replication
of the virus in b cells, followed by recruitment of macrophages to the infected islets. The
activated macrophages produce cytokines and free radicals and contribute to the destruction
of b cells, and subsequently the development of diabetes.

the residual b cells by apoptosis (FIG. 1). In contrast to EMC-D virus-induced

diabetes, KRV can cause type 1 diabetes by inducing autoimmune responses
against pancreatic b cells, rather than by direct infection of b cells. Infection
of DR-BB rats with KRV results in the breakdown of immune tolerance by the
downregulation of the Th2-type immune response, including a decrease in the
regulatory T cell population, and/or the upregulation of the Th1-type immune
response, including an increase in the autoreactive effector T cell population,
leading to autoimmune type 1 diabetes (FIG. 1).
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