This document discusses the potential role of enteroviruses, especially coxsackievirus B, in the development of type 1 diabetes mellitus (T1DM). It provides evidence from various studies that enteroviruses may infect and damage pancreatic beta cells, triggering an immune response and autoimmunity. While several viruses have been linked to T1DM, data implicates enteroviruses most strongly. The document also discusses potential mechanisms of viral persistence and the idea that enterovirus infection could initiate cycles of autoimmunity through presentation of viral and self-antigens.

1. Enterovirus
and Type1
diabetes
mellitus

2. Prepared by:
Romissaa Aly
Demonstrator of Oral medicine, Periodontology,
Diagnosis and Radiology
Al-Azhar Univerisity

3. Type 1 diabetes mellitus (T1DM) is a common
disease in which insulin-producing pancreatic β cells
are impaired by immune-mediated mechanisms that
develop as a result of a complex interaction of
genetic and environmental factors.

4. A role of environmental factors in the development
of T1DM is suggested by the continuously
increasing incidence of T1DM across the world
Gale, E. A. T (2002).
and differences in incidence of the disease in
different geographic regions.
Jaïdane, H. (2008).

5. For example, the neighboring populations of
Finland and Russian Karelia display a significant
difference in the incidence of T1DM, even though
their genetic profiles are similar.
Kondrashova, A. et al.
(2005).

6. Environmental changes are believed to affect the
penetrance of T1DM susceptibility alleles.
Key genes contributing to T1DM disease susceptibility
are located in the human leukocyte antigen (HlA) class
ii locus on chromosome 6, and other loci associated
with T1DM have been identified in the human genome.
Nejentsev, S. et al.(2007), Concannon, P. et
al(2008).

7. Viruses are candidates as infectious risk factors in
the induction of strong cellular immune responses
and in the infection and damage of β cells.
Consequently, they can cause local inflammation of
the pancreas.
Although rubella virus, mumps virus,
cytomegalovirus, retroviruses and rotaviruses have
all been linked to the development of T1DM,
evidence most strongly implicates the involvement
of enteroviruses, especially coxsackievirus B.
Richer, M. J. & Horwitz, M. S.
(2009).

9. Data from initial epidemiological studies
suggested a possible link between enteroviruses,
in particular coxsackievirus B4, and T1DM.
Jaïdane, H. & Hober, (2008).

10. Enteroviruses are frequently encountered human
pathogens with a wide variety of pathologic
effect.
they are small nonenveloped viruses (30 nm).
The icosahedric capsid is made up of 60
capsomers; one capsomer comprises four
structural proteins
(vP1, vP2, vP3 and vp4).

11. Figure 1 Enterovirus structure and replication
Hober, D. & Sauter, P. (2010) Pathogenesis of type 1 diabetes mellitus: interplay between
enterovirus and host
Nat. Rev. Endocrinol. doi:10.1038/nrendo.2010.27

14. Anti-enterovirus antibodies, as well as
enterovirus RNA, are more frequently found in
the blood of patients newly diagnosed with
T1DM than in healthy individuals .
Jaïdane, H. & Hober,
(2008).

15. An association between the presence of
coxsackievirus B RNA and elevated levels of
interferon α (IFN-α) has been found in the blood of
patients at various stages of T1DM, but not in
individuals without the disease.
Chehadeh, w. et al. (2000)

16. Boys born of mothers positive for enterovirus-
specific IGM antibodies have a fivefold increased
risk of developing T1DM as compared with boys
born of mothers negative for these antibodies.
Elfving, M. et al. (2008).

17. Enteroviruses have been found in patients with the
typical autoimmune T1DM subtype, and a role of
entero- viruses is suspected in a fulminant non
autoimmune T1DM subtype that accounts for
20% of all cases of T1DM reported in Japan .
Imagawa, A., Hanafusa, T.,
(2005).

18. Prospective studies suggest that the development of
pancreatic autoimmunity is concomitant with an
enteroviral infection.
The prevalences of both enteroviral RNA and
enteroviral-specific antibodies are greater in children
with islet autoimmunity than those without.
Hyöty, H. (2002).

19. The environmental Determinants of Diabetes in
the Young (TEDDY) is a large-scale study that
aims to improve understanding of the causes of
T1DM from birth to disease onset, and to highlight
the role of environmental factors in triggering
T1DM.

20. In the TEDDY studies, enterovirus infections
screened by nested reverse transcription PCR for
enterovirus RNA and/or anti-enterovirus IGM have
been detected during pregnancy in mothers
whose children went on to develop T1DM before
the age of 15 years.
Elfving, M. et al.
(2008).

22. IFN-α is considered a marker of virus infection. the
expression of IFN-α by β cells of patients with T1DM
(detectable by immunohistochemistry and by an
increase in IFN-α mRNA expression) has been
reported, which could be a sign of the presence of a
virus in these cells.
Foulis, A. K., Farquharson, M. A.
(1987).
Huang, X. et al. (1995).

23. Enteroviruses, especially coxsackievirus B4, have
been isolated from the pancreas of patients with
T1DM.
Dotta, F. et al. (2007).
Viral particles and the enterovirus VP1 capsid protein
have been found in β cells in pancreatic tissue from
three of six organ donors with T1DM, but not in the
pancreatic tissue of 26 organ donors without T1DM.
Dotta, F. et al. (2007).

24. IN postmortem pancreatic tissue(without
inflammatory changes in the islets) in a child who
tested positive for islet-cell auto antibodies,
enteroviral VP1 could be detected, which is in
agreement with a possible role of enteroviruses in
the early stages of the pathogenic process leading
to T1DM.
Oikarinen, M. et al. (2008).

26. A role Entero viruses in the T1DM is supported by
hygiene hypothesis is a theory that explains why
T1DM incidence is paradoxically higher in
industrialized countries where the sanitary conditions
are optimal and where enterovirus infections are less
prevalent in the population.

27. According to the hygiene hypothesis, viral infections
during childhood would protect individuals from the risk
of developing T1DM or would delay disease onset.
Bach, J. F. (2005).
Thus, an inverse correlation would exist between
T1DM incidence in a given area and the frequency of
enterovirus infections within the population.
Viskari, H. et al.(2005)

28. Figure 2 Crosstalk between viral infection, genetic background and early education of the immune
system
Beeck, A. O. d. & Eizirik, D. L. (2016) Viral infections in type 1 diabetes mellitus — why the β cells?
Nat. Rev. Neurol. doi:10.1038/nrendo.2016.30

29. Enterovirus, the immune system
andT1DM

30. Various serotypes of human enterovirus B
frequently
encountered in the environment (coxsackievirus B1,
B2, B3, B4 and B5, and echovirus 6, 7, 11, 25, 29)
can infect and damage human β cells in vitro.
Roivainen, M. (2005).

31. Enteroviruses, by the double-stranded
RNA(dsRNA) generated during their replication ,
activate the production of IFN-α and IFN-β.
Genes that encode molecules of the(MHC) are
then induced by IFN.

32. Coxsackievirus B4 activates the expression of
MHC molecules in human fetal islet cell cultures.
Coxsackievirus B induces cytokines, chemokines
and intercellular adhesion molecule 1 in human
islets in vitro, which can activate mononuclear cells
in the islets that may have a role in the
pathogenesis of T1DM.
Ylipaasto, P. et al.
(2005).

33. Figure 1 Regulation of key antiviral responses in pancreatic β cells
Beeck, A. O. d. & Eizirik, D. L. (2016) Viral infections : Candidate-gene regulated factors implicated in type 1 diabetes mellitus are framed in red and the
consequences of their modulated expression and/or activity on biological function or type 1 diabetes mellitus risk indicated. Kinases and phosphatases are
indicated by green ovals and transcription factors by grey ovals. in type 1 diabetes mellitus — why the β cells?
Nat. Rev. Neurol. doi:10.1038/nrendo.2016.30

34. Figure : Candidate-gene regulated factors implicated
in type 1 diabetes mellitus are framed in red and the
consequences of their modulated expression and/or
activity on biological function or type 1 diabetes
mellitus risk indicated. Kinases and phosphatases are
indicated by green ovals and transcription factors by
grey ovals.

35. The detection of entero-viral protein in human β
cells is accompanied by over expression of classI
MHC molecules, which could be involved in the
activation of the autoimmune processes by
enterovirus infection.
Richardson, S. J., willcox, A.,
(2009).

36. Furthermore, the infection of mice with
coxsackievirus B4 results in autoimmune diabetes
accompanied by the replication of the virus in β cells.
Yoon, J. w., Onodera, T.
(1978).
Owing to the local inflammation in the pancreas that
results from infection of β cells, enteroviruses are
strongly suspected as risk factors for T1DM.

40. A pancreatic infiltrate, called insulitis, which reflects
the immune response to β cells, is present at the
symptomatic onset of T1DM.
Studies of the pancreas in patients with T1DM
suggest that the disease has remarkable
heterogeneity.
Atkinson, M. A. & Gianani, R. (2009).

41. The detection of enteroviruses in patients newly
diagnosed with T1DM could be seen as a final step in
an ongoing autoimmune process of β-cell damage.
However, the presence of enteroviral protein (VP1) in
insulitis-free islets from a child without T1DM who was
islet-cell-autoantibody-positive open the debate about
the possible role of viruses in the early stages of the
disease.
Oikarinen, M. et al. (2008).

42. But Several groups have published data which
indicate that enteroviruses may target β cells,
which is in agreement with the hypothesis that
they may play a role in the initial disturbances of
β cells.
β-cell death might precede immune-cell
infiltration in the islets and may actually
contribute to immune infiltration.
Bortell, R., Pino, S. C., Greiner,
(2008).

43. Indeed, in human islets infected with coxsackievirus
B5, expression of pattern- recognition receptors
(such as toll-like receptor 3, RIG-1 and IFIH-1)
increases.
these receptors help the innate immune system
recognize microorganisms.

44. Neo-autoantigens could be generated by
misfolding of proteins (such as insulin) as a
result of β-cell stress.
Rossini, A.
A.(2004).
β-cell death and stress may initiate the
autoimmune attack that precedes immune-cell
infiltration in the islets.

46. Figure : Viral infections or other inflammatory stimuli
(1) will activate APCs (2) that secrete mediators that
attract lymphocytes to "rolling" and entry into the
pancreatic tissue (3). Some beta cells might be directly
destroyed via cytotoxicity (4) and others by cytokines
secreted from lymphocytes (5) or activated APCs (6).
Courtesy of Urs Christen, La Jolla Institute for Allergy
and Immunology, San Diego, CA.

48. Figure : During a localized virus infection in the pancreas. Cytokines,
such as interleukin-1β (IL-1β), IL-12, interferon-γ (IFNγ) and tumour
necrosis factor (TNF), secreted by immune cells, such as
macrophages, dendritic cells (DCs), CD4+ and CD8+ T cells and
natural killer (NK) cells, could cause direct damage (such as
apoptosis) of β-cells cells but could also induce self-defence
mechanisms;
for example, IFNγ induces indoleamine 2,3-dioxygenase (IDO)
expression by β-cells. Chemokines such as CXC-chemokine ligand 10
(CXCL10), CC-chemokine ligand 2 (CCL2) and CCL20 secreted by β-
cells recruit macrophages and other inflammatory cells to the islet
area.

49. Natural killer group 2, member D (NKG2D) expressed by NK cells
binds to retinoic acid early transcript 1 (RAE1) expressed by β-cells in
non-obese diabetic (NOD) mice, and is associated with β-cell
damage. NKp46 engagement by an unknown ligand on β-cells causes
degranulation of NK cells.
Negative co-stimulatory molecules, such as programmed cell death
ligand 1 (PDL1) expressed by β-cells can modulate diabetogenic T
cell attack.

50. Continue Figure :
β-cell antigens (such as peptides derived from insulin or pro-
insulin) can be presented by MHC class I molecules and recognized
by diabetogenic CD8+ T cells. MHC class II molecules expressed by
antigen-presenting cells such as DCs can also present β-cell
antigens (peptides such as WE14 from chromogranin A, insulin and
pro-insulin) that can induce islet antigen-specific CD4+ T cell
expansion. . FASL-expressing T cells can mediate apoptosis through
interaction with FAS expressed on β-cells.

53. Figure : Dying tissue cells release nucleic acids; these
form large complexes with antimicrobial peptides, such
as LL37 in humans and cathelin-related antimicrobial
peptide (CAMP) in mice, and with endogenous DNA-
binding proteins, such as high mobility group protein B1
(HMGB1).
These DNA and RNA complexes activate pDCs via Toll-like
receptor 9 (TLR9) and TLR7, respectively, and they induce
the production of type I IFN.

54. Fig. :In turn, type I IFN promotes T cell activation, autoantibody
production by B cells and the release of neutrophil extracellular
traps (NETs) that consist of DNA–antimicrobial peptide complexes.
Autoreactive antibodies activate neutrophils and form DNA-
containing immune complexes that are preferentially endocytosed
by pDCs via Fc receptors.
Self-nucleic acids also activate classical DCs (cDCs) and they
promote the release of inflammatory cytokines and the priming of T
cells that are specific for self antigens in a process that is also
facilitated by type I IFN.

58. Coxsackievirus B and other enteroviruses are able to
cause persistent infections.
Colbère-Garapin, F. (2002). Frisk, G., Jansson, K
(2001).
The viral persistence would result from an inappropriate
immune response to enteroviruses, as suggested by a
reduced activation and cytokine production by Tcells
from patients with T1DM in response to in vitro
challenge with coxsackievirus B4 antigens.
Skarsvik, S. et al. (2006).

59. The detection of enterovirus components in gut
mucosa of patients with T1DM suggest that the
intestine could provide a reservoir for persistent
enterovirus .
Oikarinen, M. et al. (2008).
Interestingly, enterovirus RNA has been detected in
pancreatic tissue of patients newly diagnosed with
T1DM and also in the tissue of patients with long-
standing T1DM.
Ylipaasto, P. et al. (2004).

60. T1DM might rely on cyclical processes, also
called relapsing–remitting (like those observed in
chronic viral infections). (REF. 72)
von Herrath, M., Sanda, S.
(2007).
In this process, an autoimmune response could
be raised against neoautoantigens at the same
time as cellular stress or inflammation and during
each relapse.

61. In addition, there is persistent infection (through
the presentation of viral antigens and self-
antigens) might result in the induction of an
antiviral and autoimmune response.

62. JA Bluestone et al. Nature 464, 1293-1300 (2010)
doi:10.1038/nature08933
Immunologic history of type 1 diabetes.

63. Every β-cell damage event would trigger the
recognition of other autoantigenic epitopes.
this process would increase the population of
neoantigens and epitopes displayed, and result in the
activation of autoreactive t cells, a process called
epitope spreading.
von Herrath, M., Sanda, S. & Herold, K.
(2007).

65. Alocal infection of β cells and the inflammatory
reaction that accompanies it, which causes tissue
destruction and release of sequestered self-antigens
from the islets, can induce the recruitment of auto
reactive T lymphocytes directed against these antigens
but not against viral antigens.
Horwitz, M. S., Fine, C., Ilic, A.
(2001).
Horwitz, M. S., Ilic, A., Fine, C.,
(2002).

67. A molecular mimicry has been detected between
pathogens and autoantigens recognized by
antibodies or T cells of patients with autoimmune
diseases.
Serreze, D. V., Ottendorfer, E. w.,
(2000).
Induction or acceleration of autoimmunity could be
related to infections with pathogens that have
similar structures as autoantigens.
Filippi, C. M. & von Herrath, (2008).

69. well-known model of molecular mimicry in T1DM
involves the 2C viral protease of enterovirus and
the islet auto antigen GaD65 that share a common
amino acid sequence called PEVREK.
Vreugdenhil, G. R. et al. (1998)
However, the hypothesis that this homology has a
role
in T1DM pathogenesis has been challenged and is
the subject of controversy.
Schloot, N. C. et al. (2001).

71. A defect in tolerance towards β-cell antigens at the
peripheral level is based on anomalies of regulatory
T lymphocytes (T. REG).
T.REG cells, which are a specialized subset of T cells
whose functions include the suppression of auto-
reactive T cells, play a role in the progression of
T1DM.
Filippi, C. M., Estes, E. A.,
(2009)
They maintain at least a partial tolerance to self-
antigens in the pancreas.

72. Whether viral infections have effects on T.REG cells
is not known; however, the hypothesis cannot be
excluded because toll- like receptor signaling,
provoked by viral infections, can inhibit the
suppressive activity of T.ReG cells indirectly by
causing inflammation, and possibly directly as well.
Fife, B. T. et al. (2006).

73. Thus, in addition to their possible role in
the activation of β-cell-reactive T cells,
viruses can reduce the capacity of
T.REG cells to maintain tolerance. So ,
whether enterovirus infections have such
an effect on T.REG cells should be
investigated.

76. The ADE of coxsackievirus B4 infection of
monocytes involves CAR (coxsackie virus and
adeno-virus receptor), FcγRII and FcRrIII (receptors
for the Fc portion of the IgG molecule) at the cell
surface and results in the synthesis of IFN-α by
these cells.
Hober, D., Chehadeh, w., Bouzidi, A. (2001).

78. The virus-induced synthesis of IFN-α (increased by
specific antibodies) is associated with the
development of a TH1-type immune reaction, which
can contribute to the development of autoimmune
reactions .
The Antibodies directed against VP4 or its peptides
were more prevalent and at higher levels in patients
with T1DM than in those without the disease.
Chehadeh, w. et al. (2005), Sauter, P. et al.
(2008).

79. The spreading of coxsackievirus B to β cells can,
consequently, be promoted by the ADE of the
enterovirus infection, which canfor that reason be
implied in the early steps of the immune response
against these cells.
The persistence of enteroviruses could be
associated with an increased production of enhancing
antibodies.

82. Susceptibility and resistance to T1DM are both
affected by genetic factors.
The occurrence of T1DM in monozygotic twins is
30–40%, and the risk that siblings of patients with
T1DM have of developing the disease is 6.0%
versus only 0.3% for the general population.
Jaïdane, H. & Hober, D. (2008).

83. Several loci are associated with the disease, which
suggests that its pathogenesis is polygenic.
Genes within the HlA region that encode
antigen-presenting molecules, especially the
DQ and DR loci, are associated with the risk of
T1DM.
Lowe, C. E. et al.
(2007)
In addition, some of the loci are associated with
antiviral activities.

84. OAS1 gene
a single nucleotide polymorphism exists in the OAS1
gene that is strongly associated with basal activity
of the gene product, 2'5'-oligoadenylate synthetase
1 (OAS1) a key antiviral enzyme that synthesizes
oligoadenylates that activate a latent RNase (RNaseL).
this enzyme degrades endogenous and viralRNA,
and hence impedes viral replication and promotes cell
death.

85. How would the antiviral enzyme activity of individuals
with variants of OAS1 increase their risk of T1DM ?
One explanation is that increased enzyme activity,
induced by the binding of 2'5'oas1 to intracellular
viral dsRNA, directly damages sensitive β cells (by
RNaseL-mediated degradation of cellular RNA).

86. Another explanation is that variants that confer a
low risk of T1DM produce one of the isoforms of
the protein (p48) that has a proapoptotic activity
that is independent of its synthetase activity,
Ghosh, A., Sarkar, S. N.,
(2001)
which could trigger the apoptosis of β cells infected
with enterovirus and hinder viral spread to
neighboring cells.

87. By contrast, individuals with variants that expose
them to an increased risk of T1DM may produce
little or no p48, which would reduce apoptosis-
mediated antiviral responses, resulting in
extensive β-cell damage and initiation of
autoimmune attack.
Field, L. L. et .
(2005).

88. IFIH1 is a gene that has a role in innate immunity
by the recognition of picornavirus RNA.
Shigemoto, T. et al. (2009).
The Picornaviridae family includes the
enteroviruses, such as coxsackievirus B4, which
are potential environmental triggers for T1DM.
IFIH1

89. The IFIH1 gene encodes the interferon-induced
helicase IFIH1 (also known as melanoma
differentiation associated protein 5) ,aviral RNA-
activated apoptosis protein that has widespread
expression in lymphoid and other tissues, and that
senses and triggers clearance responses in virally
infected cells.
Yoneyama, M. et al. (2005).
Meylan, E., Tschopp, J. & Karin, M.
(2006).

90. How does IFIH1 act to confer a risk of T1Dm?
following to viral infections, the transcription of
the IFIH1 gene is up regulated.
IFIH1 signaling activates IFN-regulator factors
and transcription factors, like nuclear factor κB, and
activates target genes such as those that encode
IFN-α and IFN-β and interferon- inducible genes
(Figure 2).

91. Figure 2 IFIH1-mediated β-cell response to infection with an enterovirus
Hober, D. & Sauter, P. (2010) Pathogenesis of type 1 diabetes mellitus: interplay between
enterovirus and host
Nat. Rev. Endocrinol. doi:10.1038/nrendo.2010.27

92. In response to enterovirus infections, individuals
with elevated IFIH1 levels may have an increased
stimulating capacity of dendritic cells and a
elevated production of pro-inflammatory
cytokines, which may promote the development of
T1DM.
Signal transduction pathways that involve cytoplasmic
IFIH1 considerably increase the ability of human
monocyte-derived dendritic cells to stimulate
proliferation of CD4+ T cells.
Gröschel, S. et al. (2008).

93. Figure : Regulation of key antiviral responses in pancreatic β cells
Beeck, A. O. d. & Eizirik, D. L. (2016) Viral infections in type 1 diabetes mellitus — why the β cells?
Nat. Rev. Neurol. doi:10.1038/nrendo.2016.30

95. Viruses play a dual role in T1DM. Devil or angel?
viral infections can protect individuals from the risk
of developing T1DM.
Bach, J. F. (2005).
The results of studies performed in animal
models indicate that enteroviral infections
before weaning are beneficial, whereas infections
after this period have an opposite effect.
Tracy, S. & Drescher, K. M.
(2007).

97. Together, the nature of the virus, its tropism for β
cells, and the timing of the infection have an
important role in T1DM occurrence.
Filippi, C. M. (2008).
The interplay between enterovirusinfection, β cells,
the immune system (innate and adaptive) and host
genes and the processes able to play a part are
summarized in
Figure 3.

98. Figure 3 Virus-induced pathogenesis of type 1 diabetes mellitus (T1DM)
Hober, D. & Sauter, P. (2010) Pathogenesis of type 1 diabetes mellitus: interplay between
enterovirus and host
Nat. Rev. Endocrinol. doi:10.1038/nrendo.2010.27