**2. Various viruses have been associated with the development of type 1 diabetes**

The role of viral infections in the pathogenesis of T1D has long been suspected and several viruses have been associated with T1D in various studies [160, 162]. In humans, observa‐ tions of acute diabetes succeeding to destruction of β-cells by cytopathic effect of viral infec‐ tion remain exceptional. Some viruses, as mumps, influenza B virus or human herpes virus 6 have already been reported in cases of acute T1D. Nevertheless, the fact that T1D devel‐ oped after the infection by such commun viruses suggest that factors within the host play more important roles than virus itself in the etiology of T1D [27, 59, 126].

The relationship between viral infection and T1D is mainly based on epidemiological argu‐ ments. The incidence of many allergic and autoimmune diseases has increased in developed countries (North-South gradient) over the past three decades, particularly in young chil‐ dren. Concomitantly, there was a clear decrease in the incidence of many infectious diseases in these countries, probably explained by the introduction of antibiotics, vaccination, and an improved hygiene and better socioeconomic conditions [6, 163].

Interestingly, viruses have been reported to be associated with T1D occurrence in animals. Experimental animal models, as BioBreeding (BB)-rat, nonobese diabetic (NOD) mouse or specific transgenic mouse strains, were used to investigate the mechanism by which viruses can modulate diabetogenesis.

#### **2.1. Viruses and human type 1 diabetes**

#### *2.1.1. Rubella*

Several reports have shown that congenital rubella was associated with induction of islet au‐ toantibodies in 10% to 20% of cases of congenital rubella, within 5 to 25 years [18, 56, 71]. The serum levels of antibodies against measles, mumps, and rubella (MMR) and autoanti‐ bodies against pancreas islet cells (ICA), islet cell surface, glutamic acid decarboxylase auto‐ antibodies (GADA), and insulin were determined in 386 school children between 11 and 13 years of age, before and 3 months after vaccination with combined MMR vaccine. It has been shown that children with rubella antibodies before vaccination had higher levels of ICA than seronegative children [98]. However, a study conducted in 2003 showed inconsis‐ tent results: in fact, no signs of β-cells autoimmunity (ie detection of ICA, insulin autoanti‐ bodies (IAA), antibodies to the tyrosine phosphatase related IA-2 molecule (IA-2 A) and glutamic acid decarboxylase (GADA)) were detected in 37 subjects with congenital rubella syndrome or exposed to rubella virus during fetal life [165]. The role of rubella in the trig‐ gering of T1D has been determined in hamsters. This study revealed that an autoimmune process and diabetes developed after rubella virus infection in neonatal hamsters [121]. Some authors suggested the molecular mimicry as a mechanism for rubella virus causing T1D, on the basis of co-recognition of β-cell protein determinants, such as GAD, and various rubella peptides by T-cells [118]. Recently, a clinical study has confirmed a significant asso‐ ciation between type 1 diabetes incidence and rubella in children in Italia [120].

#### *2.1.2. Rotavirus*

Interplay between immune response, genetic and environmental factors such as nutriments, drugs, toxin and viruses play a role in the pathogenesis of the disease. Several teams paid attention to the relationship between viruses and type 1 diabetes, and their role in the patho‐ genesis of the disease. A novel subtype of type 1 diabetes called fulminant type 1 diabetes, without evidence of autoimmunity has been observed [61]. In that disease the role of viruses

The relationship between type 1 diabetes in human beings and animals and various viruses belonging to different families has been investigated. Enteroviruses are among the viruses

After a presentation of the role of various viruses in the disease we will focus on enteroviruses, and then the clinical studies that were conducted to assess the relationship between enterovi‐ ruses and autoimmune T1D will be detailled. Thereafter the results of experimental investiga‐ tions aimed to elucidate the link between these viruses and the disease will be analyzed.

**2. Various viruses have been associated with the development of type 1**

The role of viral infections in the pathogenesis of T1D has long been suspected and several viruses have been associated with T1D in various studies [160, 162]. In humans, observa‐ tions of acute diabetes succeeding to destruction of β-cells by cytopathic effect of viral infec‐ tion remain exceptional. Some viruses, as mumps, influenza B virus or human herpes virus 6 have already been reported in cases of acute T1D. Nevertheless, the fact that T1D devel‐ oped after the infection by such commun viruses suggest that factors within the host play

The relationship between viral infection and T1D is mainly based on epidemiological argu‐ ments. The incidence of many allergic and autoimmune diseases has increased in developed countries (North-South gradient) over the past three decades, particularly in young chil‐ dren. Concomitantly, there was a clear decrease in the incidence of many infectious diseases in these countries, probably explained by the introduction of antibiotics, vaccination, and an

Interestingly, viruses have been reported to be associated with T1D occurrence in animals. Experimental animal models, as BioBreeding (BB)-rat, nonobese diabetic (NOD) mouse or specific transgenic mouse strains, were used to investigate the mechanism by which viruses

Several reports have shown that congenital rubella was associated with induction of islet au‐ toantibodies in 10% to 20% of cases of congenital rubella, within 5 to 25 years [18, 56, 71].

is strongly suspected as well, but is out of the scope of this chapter.

**diabetes**

26 Type 1 Diabetes

most able to be involved in the pathogenesis of autoimmune type 1diabetes.

more important roles than virus itself in the etiology of T1D [27, 59, 126].

improved hygiene and better socioeconomic conditions [6, 163].

can modulate diabetogenesis.

*2.1.1. Rubella*

**2.1. Viruses and human type 1 diabetes**

Rotavirus (RV), the most common cause of childhood gastroenteritis, has been suspected to trigger or exacerbate T1D in a few studies. Honeyman *et al.* showed a specific and highly significant association between RV seroconversion and increases in autoantibodies. Serum of 360 children with a parent or sibling with type 1 diabetes had been assayed for IAA, GA‐ DA, and IA-2A every 6 months from birth. In all children, 24 children had been classified as high-risk children because they developed diabetes or had at least 2 islet antibodies or 1 islet antibody detected on at least 2 occasions within the study period. In high-risk children, 86% developed antibodies to IA-2, 62% developed insulin autoantibodies, and 50% developed antibodies to GAD in association with first appearance or increase in RV IgG or IgA [70]. In 2002, Coulson et al demonstrated that rotavirus could infect pancreas *in vivo* [35]. In this study, nonobese diabetic (NOD) mice were shown to be susceptible to rhesus rotavirus in‐ fection. Pancreatic islets from NOD mice, nonobese diabetes-resistant mice, fetal pigs, and macaque monkeys supported various degrees of rotavirus growth. Human rotaviruses that were propagated in African green monkey kidney epithelial (MA104) cells in the presence of trypsin as previously described [128] replicated in monkey islets only [35]. In another study, the effect of RV infection on diabetes development, once diabetes was established, was de‐ termined on NOD and NOD8.3 TCR (transgenic for a T-cell receptor (TCR)) mice. The de‐ gree of diabetes acceleration was related to the serum antibody titer to RV. Thus, rotavirus infection aggravated insulitis and exacerbated diabetes, after β-cell autoimmunity was es‐ tablished [60]. Furthermore, rotavirus was also suspected to contain peptide sequences, in VP7 (viral protein 7), highly similar to T-cell epitopes in the islet autoantigens GAD and ty‐ rosine phosphatase IA-2, suggesting that T-cells directed against RV could induce or ampli‐ fy islet autoimmunity by molecular mimicry, in children with genetic susceptibility. Honeyman *et al.* also demonstrated that peptides in RV-VP7, similar to T-cell epitopes in IA-2 and GAD65, bound strongly to HLA-DRB1\*04. The proliferative responses of T-cells to rotavirus peptide and islet autoantigen-derived peptides were significantly correlated [72]. Altogether, these observations suggested that RV infection could trigger or exacerbate islet autoimmunity by molecular mimicry.

K18 variant, which is transcriptionally silent, could be directly transactivated by EBV (Epstein Barr Virus) or HHV-6 (human herpes virus 6), or alternatively through the EBV or

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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29

Rubella virus, rotavirus, mumps virus and endogenous retroviruses are RNA viruses whose role in type 1 diabetes has been suspected. In addition to RNA viruses, it has been reported that DNA viruses as well could be involved in the development of the disease as described

In 1988, Numazaki et al showed that cytomegalovirus (CMV) was able to infect tissue mon‐ olayer cultures of human fetal islets [112]. CMV infection apparently did not cause direct destruction of β-cells but was leading to changes in production of insulin [112]. Hille‐ brands *et al.* demonstrated that R(at)-CMV accelerated onset of diabetes without infecting pancreatic islets in BB-rats and suggested that virus-induced recruitment of peritoneal mac‐ rophages to the pancreas triggered the accelerated development of insulitis by enhancing activation of T-cells in pancreas [65]. In 2003, van der Werf et al indicated that R-CMV in‐ duced a very strong T-cell proliferative response in BB-rats suggesting that R-CMV might directly activate autoreactive T-cells resulting in accelerated onset of diabetes [161]. In 2010, Smelt et al demonstrated that RCMV induced a low, persistent infection in rat β-cells, as‐ sociated with an increasing β-cell immunogenicity, which might be an essential step in βcells destruction and in the development or the acceleration of the onset of T1D [137]. Concerning the role of Human CMV (HCMV) in diabetogenesis, [64] postulated that there is T-cell cross-reactivity between Human CMV (HCMV) and GAD65 in pancreatic islet βcell. HCMV-derived epitope could be naturally processed by dendritic cells and recog‐ nized by GAD65 reactive T-cells. Thus, HCMV may be involved in the loss of T-cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity [64]. In 2008, Aarnisalo et al analysed specific anti-CMV IgG antibodies in 169 serum sam‐ ples from children who had developed the first T1D-associated autoantibody by the age of 2 years, and, in parallel, in 791 serum control from healthy children [1]. No associa‐ tion between perinatal CMV infection and progression to T1D was observed. This study con‐ cluded that perinatal CMV infections were not particularly associated with early serological signs of beta cell autoimmunity or progression to T1D in children with diabetes risk-asso‐ ciated HLA genotype [1]. However, serological, immunological, histological signs of auto‐ immunity and allograft rejection appeared concomitantly with early CMV infections in one type 1 diabetic patient receiving pancreas allograft. This observation suggests that persis‐ tent CMV infections might be relevant to the pathogenesis of type 1 diabetes [177].

Several cases of autoimmune disease occurrence after an acute infection with parvovirus B19 have been reported. Kasuga *et al.* reported a case of a young adult who developed new onset T1D after an infection with parvovirus B19. Serum levels of B19 IgM and antibodies to the diabetic autoantigen IA-2 were significantly elevated. The authors noted homology in amino acid sequences between B19 and the extracellular domain of IA-2 [88, 113]. Munakata

HHV-6- induced production of the IFN-α [143, 144].

in the following paragraphs.

*2.1.5. Cytomegalovirus*

*2.1.6. Parvovirus B19*

#### *2.1.3. Mumps*

In 1992, Parkkonen et al showed that mumps virus was able to infect β-cells, leading to a minor decrease in insulin secretion in human fetal islet cultures [119]. The infection was in‐ variably associated with an increase in the expression of human leucocyte antigen (HLA) class I molecules, mediated by soluble factors secreted by infected T cells, which could exag‐ gerate the autoimmune process in pre-diabetic individuals by increasing the activity of au‐ toreactive cytotoxic T cells [119]. Moreover, ICA have been observed in 14 out of 30 sera of children with mumps. In most children, the ICA persisted for no more than 2-4 months, al‐ though 2 children have been positive for 15 months. Nevertheless, no ICA-positive child ac‐ quired diabetic glucose metabolism, apart from one child who had persistent ICA and acquired diabetes mellitus three weeks after mumps infection [62]. Since the introduction of vaccination against MMR in most of occidental countries, several studies have reported on the relation between vaccination at childhood and the development of T1D [41, 78, 79]. Hyo‐ ty *et al.* demonstrated that vaccination against MMR in Finland was followed by a plateau in the rising incidence of T1D 6–8 years later suggesting a causal relation between these viral infections and the development of T1D [79]. However, the incidence of T1D continued to rise after the plateau. Other studies hypothesized that childhood vaccination would rather promote the development of T1D. No evidence has been found for the triggering effect of childhood vaccination on the development of T1D later in life [41, 78]. Hyoty et al. described a shared epitope, a 7 amino acid-long sequence (YQQQGRL), in mumps virus nucleocapsid and in MHC class II-associated invariant chain, which might cause immunological cross-re‐ activity between these molecules [80].

#### *2.1.4. Human Endogenous Retroviruses*

Human Endogenous Retroviruses (HERVs) are sequences which occupy about 10% of the human genome and are thought to be derived from ancient viral infections of germ cells. In some medical conditions, HERVs genes could be transcripted, expressed in protein and could be responsible of the development of autoantibodies that might react against host pro‐ teins. As a result, these mechanisms could lead to autoimmune diseases, such as T1D. HERVs may also dysregulate the immune response by being moved and inserted next to certain genes involved in immune regulation whose expression would be consequentially altered. Finally, HERVs are known to induce proinflammatory cytokines production, as IL-1β, IL-6, or TNF-α, by cells, such as monocytes [10]. The HERV-K18 variant has been shown to encode for a superantigen (SAg) that is recognized by T-cells with TCR Vβ7 chains and causes dysregulation of the immune system. HERV-K18 mRNA has been found to be enriched in tissues of patients with acute T1D. HERV-K18 transcription and SAg function in cells capable of efficient presentation are induced by proinflammatory stimuli and may trig‐ ger progression of disease to insulitis or from insulitis to overt diabetes [101]. The HERV- K18 variant, which is transcriptionally silent, could be directly transactivated by EBV (Epstein Barr Virus) or HHV-6 (human herpes virus 6), or alternatively through the EBV or HHV-6- induced production of the IFN-α [143, 144].

Rubella virus, rotavirus, mumps virus and endogenous retroviruses are RNA viruses whose role in type 1 diabetes has been suspected. In addition to RNA viruses, it has been reported that DNA viruses as well could be involved in the development of the disease as described in the following paragraphs.

### *2.1.5. Cytomegalovirus*

rotavirus peptide and islet autoantigen-derived peptides were significantly correlated [72]. Altogether, these observations suggested that RV infection could trigger or exacerbate islet

In 1992, Parkkonen et al showed that mumps virus was able to infect β-cells, leading to a minor decrease in insulin secretion in human fetal islet cultures [119]. The infection was in‐ variably associated with an increase in the expression of human leucocyte antigen (HLA) class I molecules, mediated by soluble factors secreted by infected T cells, which could exag‐ gerate the autoimmune process in pre-diabetic individuals by increasing the activity of au‐ toreactive cytotoxic T cells [119]. Moreover, ICA have been observed in 14 out of 30 sera of children with mumps. In most children, the ICA persisted for no more than 2-4 months, al‐ though 2 children have been positive for 15 months. Nevertheless, no ICA-positive child ac‐ quired diabetic glucose metabolism, apart from one child who had persistent ICA and acquired diabetes mellitus three weeks after mumps infection [62]. Since the introduction of vaccination against MMR in most of occidental countries, several studies have reported on the relation between vaccination at childhood and the development of T1D [41, 78, 79]. Hyo‐ ty *et al.* demonstrated that vaccination against MMR in Finland was followed by a plateau in the rising incidence of T1D 6–8 years later suggesting a causal relation between these viral infections and the development of T1D [79]. However, the incidence of T1D continued to rise after the plateau. Other studies hypothesized that childhood vaccination would rather promote the development of T1D. No evidence has been found for the triggering effect of childhood vaccination on the development of T1D later in life [41, 78]. Hyoty et al. described a shared epitope, a 7 amino acid-long sequence (YQQQGRL), in mumps virus nucleocapsid and in MHC class II-associated invariant chain, which might cause immunological cross-re‐

Human Endogenous Retroviruses (HERVs) are sequences which occupy about 10% of the human genome and are thought to be derived from ancient viral infections of germ cells. In some medical conditions, HERVs genes could be transcripted, expressed in protein and could be responsible of the development of autoantibodies that might react against host pro‐ teins. As a result, these mechanisms could lead to autoimmune diseases, such as T1D. HERVs may also dysregulate the immune response by being moved and inserted next to certain genes involved in immune regulation whose expression would be consequentially altered. Finally, HERVs are known to induce proinflammatory cytokines production, as IL-1β, IL-6, or TNF-α, by cells, such as monocytes [10]. The HERV-K18 variant has been shown to encode for a superantigen (SAg) that is recognized by T-cells with TCR Vβ7 chains and causes dysregulation of the immune system. HERV-K18 mRNA has been found to be enriched in tissues of patients with acute T1D. HERV-K18 transcription and SAg function in cells capable of efficient presentation are induced by proinflammatory stimuli and may trig‐ ger progression of disease to insulitis or from insulitis to overt diabetes [101]. The HERV-

autoimmunity by molecular mimicry.

activity between these molecules [80].

*2.1.4. Human Endogenous Retroviruses*

*2.1.3. Mumps*

28 Type 1 Diabetes

In 1988, Numazaki et al showed that cytomegalovirus (CMV) was able to infect tissue mon‐ olayer cultures of human fetal islets [112]. CMV infection apparently did not cause direct destruction of β-cells but was leading to changes in production of insulin [112]. Hille‐ brands *et al.* demonstrated that R(at)-CMV accelerated onset of diabetes without infecting pancreatic islets in BB-rats and suggested that virus-induced recruitment of peritoneal mac‐ rophages to the pancreas triggered the accelerated development of insulitis by enhancing activation of T-cells in pancreas [65]. In 2003, van der Werf et al indicated that R-CMV in‐ duced a very strong T-cell proliferative response in BB-rats suggesting that R-CMV might directly activate autoreactive T-cells resulting in accelerated onset of diabetes [161]. In 2010, Smelt et al demonstrated that RCMV induced a low, persistent infection in rat β-cells, as‐ sociated with an increasing β-cell immunogenicity, which might be an essential step in βcells destruction and in the development or the acceleration of the onset of T1D [137]. Concerning the role of Human CMV (HCMV) in diabetogenesis, [64] postulated that there is T-cell cross-reactivity between Human CMV (HCMV) and GAD65 in pancreatic islet βcell. HCMV-derived epitope could be naturally processed by dendritic cells and recog‐ nized by GAD65 reactive T-cells. Thus, HCMV may be involved in the loss of T-cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity [64]. In 2008, Aarnisalo et al analysed specific anti-CMV IgG antibodies in 169 serum sam‐ ples from children who had developed the first T1D-associated autoantibody by the age of 2 years, and, in parallel, in 791 serum control from healthy children [1]. No associa‐ tion between perinatal CMV infection and progression to T1D was observed. This study con‐ cluded that perinatal CMV infections were not particularly associated with early serological signs of beta cell autoimmunity or progression to T1D in children with diabetes risk-asso‐ ciated HLA genotype [1]. However, serological, immunological, histological signs of auto‐ immunity and allograft rejection appeared concomitantly with early CMV infections in one type 1 diabetic patient receiving pancreas allograft. This observation suggests that persis‐ tent CMV infections might be relevant to the pathogenesis of type 1 diabetes [177].

#### *2.1.6. Parvovirus B19*

Several cases of autoimmune disease occurrence after an acute infection with parvovirus B19 have been reported. Kasuga *et al.* reported a case of a young adult who developed new onset T1D after an infection with parvovirus B19. Serum levels of B19 IgM and antibodies to the diabetic autoantigen IA-2 were significantly elevated. The authors noted homology in amino acid sequences between B19 and the extracellular domain of IA-2 [88, 113]. Munakata et al described the case of a 40-year-old Japanese woman, in which three autoimmune dis‐ eases occurred after acute parvovirus B19 infection: rheumatoid arthritis, T1D and Graves'disease [106]. Some authors attempted to explain these observations. Parvovirus B19 is known to promote a T-cell-mediated lymphoproliferative response, through the presenta‐ tion by HLA class II antigen to CD4 cells and thus could theoretically generate T-cell-medi‐ ated autoimmunity [166]. Vigeant et al suggested that parvovirus B19 infection may lead to chronic modulation of the autoimmune response in predisposed individuals [164].

of Mda5 developed transient hyperglycemia when infected with EMCV-D. Thus, in the case of EMCV-D which infects and damages directly the pancreatic β cells, optimal functioning

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31

Ellerman et al. demonstrated the ability of Kilham rat virus (KRV), an environmentally ubiq‐ uitous rat parvovirus, to precipitate autoimmune diabetes in BioBreeding Diabetes-Resistant (BBDR) rats that were not susceptible to spontaneous diabetes [47]. Chung et al. showed the important role of macrophages and macrophage-derived cytokines (IL-12, TNF-α, and IL-1β) in the KRV-induced autoimmune diabetes in the BBDR rats [29]. As it had been previ‐ ously shown, KRV did not directly infect β-cells. Thus, Choung et al. investigated the proc‐ ess by which KRV induced autoimmune pancreatic cells destruction. They discovered that it was rather due to a disrupted immune balance: Th1-like CD45RC+CD4+ and cytotoxic CD8+ T-cells were up-regulated whereas Th2-like CD45RC-CD4+ T-cells were down-regulated. Thus, KRV might be responsible for the activation of autoreactive T cells that are cytotoxic to beta cells, resulting in T cell-mediated autoimmune diabetes. In the same study, this group demonstrated that KRV-induced autoimmune diabetes in BBDR rats was not due to molecular mimicry [30]. Zipris et al. reported that infection by KRV or H-1, a close homo‐ logue virus of KRV, induced similar humoral and cellular immune responses in BBDR rats and Wistar Furth (WF) rats. Nevertheless, only KRV induced a decrease in splenic CD4+CD25+ T cells (regulatory T cells or Treg) able to suppress autoreactivity, in both rat strains. KRV was able to induce diabetes in BBDR rats but not in WF rats. The disease was associated with accumulation of non proliferating Treg in pancreatic lymph nodes. Together these data suggest a virus- and rat strain- specific mechanism of KRV-induced diabetes in genetically susceptible rats as BBDR rats, through an alteration of T cell regulation. It ap‐ pears that Treg are no longer able to inhibit autoreactive T cells activation [178]. It has also been shown that proinflammatory cytokines IL-6 and IL-12p40 were producted by spleen cells cultured in vitro in the presence of KRV in BBDR and WF rats. Ligation of TLR9 with CpG DNA induced the same pattern of cytokine production. In response to both KRV and CpG DNA, spleen cell populations enriched for B cells (CD45R+) secreted significantly more IL-12p40 than populations enriched for non B-cells (CD45R-). KRV was also able to stimu‐ late Flt-3L bone marrow-derived dendritic cells (DCs) to produce IL-12p40 in vitro. More‐ over, genomic DNA isolated from KRV, which is a single-strand DNA, induced the production of IL-12p40 in spleen cells from BBDR rats. Thus, the ligand within KRV that in‐ duces IL-12p40 secretion in spleen cells is viral DNA. Using appropriate inhibitors of TLRsignaling pathways, Zipris et al. indicated that the cytokine production by splenic cells was Protein Kinase R (PKR) and NF-κB dependent, whose activation leads to type I IFN produc‐ tion. KRV-induced secretion of IL-12p40 by BBDR spleen cells was inhibited by specific TLR9 inhibitors, as iCpG, and by chloroquine, which is a known inhibitor of endosomal acidification, essential step for the recruitment of TLR9 in the lysosomal compartment. Moreover, genomic DNA isolated from KRV induced the production of IL-12p40 in Flt-3Linduced DCs derived from wild-type BBDR rats but not TLR9-deficient mice. Finally, ad‐ ministration of chloroquine to virus-infected BBDR rats decreased the incidence of diabetes

of viral sensors and type 1 IFN responses are required to prevent diabetes [102].

*2.2.2. Kilham rat virus*

Although correlations between T1D and the occurrence of a viral infection that precedes the development of the autoimmune disease have been recognized, mechanisms by which vi‐ ruses activate diabetogenic processes are still elusive and difficult to prove in humans. Stud‐ ies of animal virus-induced T1D provide a lot of information concerning the possible role of virus infections in the induction of TID.

#### **2.2. Viruses and animal type 1 diabetes**

#### *2.2.1. Encephalomyocarditis virus*

A number of studies provide clear evidence that encephalomyocarditis virus (EMCV), be‐ longing to the Cardiovirus genus of the Picornaviridae family, is able to induce very rapid onset of diabetes in mice. Based upon this evidence, EMCV-induced diabetes model has been proposed as a model of fulminant T1D [135]. Several studies determined the existence of two main variants of EMCV: the nondiabetogenic variant EMC-B virus, and the diabeto‐ genic variant EMC-D virus. EMCV-D has preferential tropism for pancreatic β-cells and can induce diabetes in selective mouse strains, such as DBA/2 [102]. Nucleotide sequence analy‐ sis showed that EMC-D virus (7829 bases) differs from EMC-B virus (7825 bases) by only 14 nucleotides: two deletions of 5 nucleotides, 1 base insertion, and 8 point mutations. Further studies revealed that only the 776th amino acid, alanine (Ala-776), of the EMC virus poly‐ protein, located at position 152 of the major capsid protein VP1, is common to all diabeto‐ genic variants. In contrast, threonine in this position (Thr-776) is common to all nondiabetogenic variants [176]. A single point mutation at nucleotide position 3155 or 3156 of the recombinant EMC viral genome, resulting in an amino acid change (Ala-776 in Thr-776), leads to the gain or loss of viral diabetogenicity [84]. A three-dimensional molecu‐ lar modeling of the binding site of the EMC viral capsid protein VP1 revealed that the sur‐ face areas surrounding alanine (or glycine) at position 152 of the VP1 was more accessible, thus increasing the availability of the binding sites for attachment to β-cell receptors, result‐ ing in viral infection and the development of diabetes [85]. Baek et al. showed that macro‐ phages, especially mac-2 positive macrophages, were rapidly recruited in pancreas at the early stage of EMC-D virus infection, playing a central role in the process of pancreatic islets destruction in SJL/J mice [8, 9]. Recently, Mc Cartney et al. found that melanoma differentia‐ tion associated gene-5 (MDA5), a sensor of viral RNA eliciting IFN-I responses, IFN-α, and Toll-Like Receptor 3 (TLR3) were both required to prevent diabetes in mice infected with EMCV-D. In Tlr3-/- mice, a diabetes occured due to impaired tpe 1 IFN responses and β cell damage induced directly by virus, rather than autoimmune T cells. Mice lacking just 1 copy of Mda5 developed transient hyperglycemia when infected with EMCV-D. Thus, in the case of EMCV-D which infects and damages directly the pancreatic β cells, optimal functioning of viral sensors and type 1 IFN responses are required to prevent diabetes [102].

#### *2.2.2. Kilham rat virus*

et al described the case of a 40-year-old Japanese woman, in which three autoimmune dis‐ eases occurred after acute parvovirus B19 infection: rheumatoid arthritis, T1D and Graves'disease [106]. Some authors attempted to explain these observations. Parvovirus B19 is known to promote a T-cell-mediated lymphoproliferative response, through the presenta‐ tion by HLA class II antigen to CD4 cells and thus could theoretically generate T-cell-medi‐ ated autoimmunity [166]. Vigeant et al suggested that parvovirus B19 infection may lead to

Although correlations between T1D and the occurrence of a viral infection that precedes the development of the autoimmune disease have been recognized, mechanisms by which vi‐ ruses activate diabetogenic processes are still elusive and difficult to prove in humans. Stud‐ ies of animal virus-induced T1D provide a lot of information concerning the possible role of

A number of studies provide clear evidence that encephalomyocarditis virus (EMCV), be‐ longing to the Cardiovirus genus of the Picornaviridae family, is able to induce very rapid onset of diabetes in mice. Based upon this evidence, EMCV-induced diabetes model has been proposed as a model of fulminant T1D [135]. Several studies determined the existence of two main variants of EMCV: the nondiabetogenic variant EMC-B virus, and the diabeto‐ genic variant EMC-D virus. EMCV-D has preferential tropism for pancreatic β-cells and can induce diabetes in selective mouse strains, such as DBA/2 [102]. Nucleotide sequence analy‐ sis showed that EMC-D virus (7829 bases) differs from EMC-B virus (7825 bases) by only 14 nucleotides: two deletions of 5 nucleotides, 1 base insertion, and 8 point mutations. Further studies revealed that only the 776th amino acid, alanine (Ala-776), of the EMC virus poly‐ protein, located at position 152 of the major capsid protein VP1, is common to all diabeto‐ genic variants. In contrast, threonine in this position (Thr-776) is common to all nondiabetogenic variants [176]. A single point mutation at nucleotide position 3155 or 3156 of the recombinant EMC viral genome, resulting in an amino acid change (Ala-776 in Thr-776), leads to the gain or loss of viral diabetogenicity [84]. A three-dimensional molecu‐ lar modeling of the binding site of the EMC viral capsid protein VP1 revealed that the sur‐ face areas surrounding alanine (or glycine) at position 152 of the VP1 was more accessible, thus increasing the availability of the binding sites for attachment to β-cell receptors, result‐ ing in viral infection and the development of diabetes [85]. Baek et al. showed that macro‐ phages, especially mac-2 positive macrophages, were rapidly recruited in pancreas at the early stage of EMC-D virus infection, playing a central role in the process of pancreatic islets destruction in SJL/J mice [8, 9]. Recently, Mc Cartney et al. found that melanoma differentia‐ tion associated gene-5 (MDA5), a sensor of viral RNA eliciting IFN-I responses, IFN-α, and Toll-Like Receptor 3 (TLR3) were both required to prevent diabetes in mice infected with EMCV-D. In Tlr3-/- mice, a diabetes occured due to impaired tpe 1 IFN responses and β cell damage induced directly by virus, rather than autoimmune T cells. Mice lacking just 1 copy

chronic modulation of the autoimmune response in predisposed individuals [164].

virus infections in the induction of TID.

**2.2. Viruses and animal type 1 diabetes**

*2.2.1. Encephalomyocarditis virus*

30 Type 1 Diabetes

Ellerman et al. demonstrated the ability of Kilham rat virus (KRV), an environmentally ubiq‐ uitous rat parvovirus, to precipitate autoimmune diabetes in BioBreeding Diabetes-Resistant (BBDR) rats that were not susceptible to spontaneous diabetes [47]. Chung et al. showed the important role of macrophages and macrophage-derived cytokines (IL-12, TNF-α, and IL-1β) in the KRV-induced autoimmune diabetes in the BBDR rats [29]. As it had been previ‐ ously shown, KRV did not directly infect β-cells. Thus, Choung et al. investigated the proc‐ ess by which KRV induced autoimmune pancreatic cells destruction. They discovered that it was rather due to a disrupted immune balance: Th1-like CD45RC+CD4+ and cytotoxic CD8+ T-cells were up-regulated whereas Th2-like CD45RC-CD4+ T-cells were down-regulated. Thus, KRV might be responsible for the activation of autoreactive T cells that are cytotoxic to beta cells, resulting in T cell-mediated autoimmune diabetes. In the same study, this group demonstrated that KRV-induced autoimmune diabetes in BBDR rats was not due to molecular mimicry [30]. Zipris et al. reported that infection by KRV or H-1, a close homo‐ logue virus of KRV, induced similar humoral and cellular immune responses in BBDR rats and Wistar Furth (WF) rats. Nevertheless, only KRV induced a decrease in splenic CD4+CD25+ T cells (regulatory T cells or Treg) able to suppress autoreactivity, in both rat strains. KRV was able to induce diabetes in BBDR rats but not in WF rats. The disease was associated with accumulation of non proliferating Treg in pancreatic lymph nodes. Together these data suggest a virus- and rat strain- specific mechanism of KRV-induced diabetes in genetically susceptible rats as BBDR rats, through an alteration of T cell regulation. It ap‐ pears that Treg are no longer able to inhibit autoreactive T cells activation [178]. It has also been shown that proinflammatory cytokines IL-6 and IL-12p40 were producted by spleen cells cultured in vitro in the presence of KRV in BBDR and WF rats. Ligation of TLR9 with CpG DNA induced the same pattern of cytokine production. In response to both KRV and CpG DNA, spleen cell populations enriched for B cells (CD45R+) secreted significantly more IL-12p40 than populations enriched for non B-cells (CD45R-). KRV was also able to stimu‐ late Flt-3L bone marrow-derived dendritic cells (DCs) to produce IL-12p40 in vitro. More‐ over, genomic DNA isolated from KRV, which is a single-strand DNA, induced the production of IL-12p40 in spleen cells from BBDR rats. Thus, the ligand within KRV that in‐ duces IL-12p40 secretion in spleen cells is viral DNA. Using appropriate inhibitors of TLRsignaling pathways, Zipris et al. indicated that the cytokine production by splenic cells was Protein Kinase R (PKR) and NF-κB dependent, whose activation leads to type I IFN produc‐ tion. KRV-induced secretion of IL-12p40 by BBDR spleen cells was inhibited by specific TLR9 inhibitors, as iCpG, and by chloroquine, which is a known inhibitor of endosomal acidification, essential step for the recruitment of TLR9 in the lysosomal compartment. Moreover, genomic DNA isolated from KRV induced the production of IL-12p40 in Flt-3Linduced DCs derived from wild-type BBDR rats but not TLR9-deficient mice. Finally, ad‐ ministration of chloroquine to virus-infected BBDR rats decreased the incidence of diabetes and decreased blood levels of IL-12p40. These data indicates that the TLR9 -signaling path‐ way is implicated in the KRV-induced innate immune activation and participates to the de‐ velopment of autoimmune diabetes in the BBDR rat [179, 13].

"Environmental Triggers of Type 1 Diabetes: The MIDIA study", stool samples from 27 chil‐ dren who developed islet autoimmunity (repeatedly positive for two or three autoantibod‐ ies) and 53 children matched for age and community of residence (control group) were analyzed for human parechovirus using a semi-quantitative real-time polymerase chain re‐ action every month from the 3rd to the 35th month. Sera of children were tested for autoan‐ tibodies against GAD, IA-2, and insulin every 3 months until the age of 1 year and every 12 months thereafter. There was no significant difference in the number of infection episodes between the two groups. There was also no significant difference in the prevalence of hu‐ man parechovirus in stool samples throughout the study period, except in samples collected 3 months prior to seroconversion, in which 16/77 samples (20.8%) from cases had an infec‐

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

http://dx.doi.org/10.5772/52087

33

tion as opposed to 16/182 (8.8%) samples from controls (OR = 3.17, p = 0.022) [148].

be focused on these viruses.

(in red).

Human type 1 diabetes *Togaviridae*

Animal type 1 diabetes Encephalomyocarditis virus

Rubella virus *Paramyxoviridae* Mumps virus *Picornaviridae* Parechovirus Enterovirus

Ljungan virus

**Table 1.** Viruses involved in human and animal type 1 diabetes grouped according to their genome and their family

Various viruses were reported to be associated with human T1D: rubella and mumps virus, rotavirus, retrovirus, human parechovirus, cytomegalovirus and parvovirus B19 (table 1). In addition, viruses were reported to be associated with animal T1D: EMCV, KRV and LV, the role of which in human type 1 diabetes has also been studied (figure 2). Using animal mod‐ els, as BB-rats, NOD mice or specific transgenic mouse strains, studies suggested different mechanisms by which viruses may be involved in the initiation or modulation of autoim‐ mune process. These models suggested that a direct infection of islets, responsible for the release of autoantigens, could explain the activation of T-cells and the development of auto‐ immunity. Another hypothesis supported by some studies was the concept of molecular mimicry between virus and β-cells: a normal immune response against a viral antigen would become pathogenic for β cells due to the existence of structural homologies with the pancreatic antigen. In addition to their possible role in the activation of β-cell-reactive T cells, viruses can reduce the capacity of Treg cells to maintain tolerance. Together, these studies suggest that viruses through diffent mechanisms may trigger T1D and/or may par‐ ticipate in the amplification of the autoimmune process. In addition to the viruses presented in this section, the major candidates are enteroviruses. Therefore the rest of this review will

**RNA virus DNA virus**

*Reoviridae* Rotavirus *Retroviridae* HERVs

*Herpesviridae* Cytomegalovirus *Parvoviridae* Parvovirus B19

Kilham rat virus

EMC and KRV are natural viral pathogens of rodent that brought a lot of information as far as the virus–induced pathogenesis of T1D. The role of these viruses in the human T1D has not been reported, however, the Ljungan virus is another rodent virus that has been suspect‐ ed to be involved in human type 1diabetes.
