*4.1.1. Whole blood*

The presence of enteroviral RNA in whole blood of adult patients with T1D has been report‐ ed by our group [5]. Viral RNA was detected by RT-PCR in 42% (5/12) of patients with new‐ ly diagnosed T1D (p <0.01 vs healthy subjects) and in 8% (1/12) of previously diagnosed T1D patients suffering from metabolic ketosis decompensation (P=0.07 vs patients with newly di‐ agnosed T1D). RT-PCR was negative in the group of healthy subjects and patients with type 2 diabetes. Sequencing of amplified cDNA displayed that circulating enteroviral RNA in these patients had strong homologies with CVB (CVB3 in 4 patients with newly diagnosed T1D, CVB4 in another one, and in one previously diagnosed patient). This study demon‐ strated that enteroviral RNA could be detected in blood of adult patients at the onset or in the course of T1D.

An other study, performed also by our group, encompassed 56 patients with T1D (25 chil‐ dren whose average age was 13 years and 31 adults with an average age of 37 years), and 37 control subjects divided into 2 groups: the first comprising 24 subjects without any infec‐ tious, immunological or metabolic disease, the second group includes 13 patients with T2D [23]. The presence of IFN-α mRNA was detected by RT-PCR in whole blood of 42 out of 56 patients (75%) but in none of controls, and IFN-α was detected by a sensitive immunoassay in serum of 39 out of 56 patients. Enteroviruses-RNA sequences were detected in 50% (21/42) of patients with IFN-α in their blood, but not in patients without any IFN-α in their blood. The detection of enteroviral RNA was positive in 25% (3/12) of children with newly diagnosed T1D, 30% (4/13) of children with previously diagnosed T1D, 50% (10/20) of adult patients with newly diagnosed T1D and 36% (4/11) of adult patients with previously diag‐ nosed T1D. Sequencing of amplified cDNA displayed that circulating enteroviral RNA in these 21 patients had strong homologies with CVB (CVB3 in 8 patients; CVB4 in 8 patients; CVB2 in 5 patients). The results of sequencing of circulating enteroviral RNA were concord‐ ant with the results of anti-CVB neutralizing antibodies assay. Otherwise, there was no sig‐ nificant relationship between enterovirus detection and age of patients or the pattern of disease (metabolic decompensation or not)

In Sweden, blood spots are routinely taken on days 2-4 of life for analysis of inherited meta‐ bolic diseases in all newborns and are stored in a biobank. From this biobank, a Swedish study investigated enteroviral RNA in blood spots from 600 children in the Swedish child‐ hood diabetes register [39]. Six hundred healthy children were included as controls. Viral RNA was found in 27 out of 600 (4.5%) diabetic children compared to 14 out of 600 (2.3%) control children (p=0.04).

### *4.1.2. Serum and plasma*

enteroviral infection and T1D. Theses studies have used different techniques to detect enter‐ oviruses or their components (RT-PCR, cell culture, immunohistochemistry, in situ hybridi‐ zation...) in blood (serum, plasma and leucocytes), stools, pancreas, intestine. Several studies throughout the world have displayed a relationship between enterovirus infection and the

We will present the detection of enteroviruses and/or their components in various biological samples in patients with clinical type 1 diabetes first, and thereafter in patients with signs of

The presence of enteroviral RNA in whole blood of adult patients with T1D has been report‐ ed by our group [5]. Viral RNA was detected by RT-PCR in 42% (5/12) of patients with new‐ ly diagnosed T1D (p <0.01 vs healthy subjects) and in 8% (1/12) of previously diagnosed T1D patients suffering from metabolic ketosis decompensation (P=0.07 vs patients with newly di‐ agnosed T1D). RT-PCR was negative in the group of healthy subjects and patients with type 2 diabetes. Sequencing of amplified cDNA displayed that circulating enteroviral RNA in these patients had strong homologies with CVB (CVB3 in 4 patients with newly diagnosed T1D, CVB4 in another one, and in one previously diagnosed patient). This study demon‐ strated that enteroviral RNA could be detected in blood of adult patients at the onset or in

An other study, performed also by our group, encompassed 56 patients with T1D (25 chil‐ dren whose average age was 13 years and 31 adults with an average age of 37 years), and 37 control subjects divided into 2 groups: the first comprising 24 subjects without any infec‐ tious, immunological or metabolic disease, the second group includes 13 patients with T2D [23]. The presence of IFN-α mRNA was detected by RT-PCR in whole blood of 42 out of 56 patients (75%) but in none of controls, and IFN-α was detected by a sensitive immunoassay in serum of 39 out of 56 patients. Enteroviruses-RNA sequences were detected in 50% (21/42) of patients with IFN-α in their blood, but not in patients without any IFN-α in their blood. The detection of enteroviral RNA was positive in 25% (3/12) of children with newly diagnosed T1D, 30% (4/13) of children with previously diagnosed T1D, 50% (10/20) of adult patients with newly diagnosed T1D and 36% (4/11) of adult patients with previously diag‐ nosed T1D. Sequencing of amplified cDNA displayed that circulating enteroviral RNA in these 21 patients had strong homologies with CVB (CVB3 in 8 patients; CVB4 in 8 patients; CVB2 in 5 patients). The results of sequencing of circulating enteroviral RNA were concord‐ ant with the results of anti-CVB neutralizing antibodies assay. Otherwise, there was no sig‐ nificant relationship between enterovirus detection and age of patients or the pattern of

In Sweden, blood spots are routinely taken on days 2-4 of life for analysis of inherited meta‐ bolic diseases in all newborns and are stored in a biobank. From this biobank, a Swedish study investigated enteroviral RNA in blood spots from 600 children in the Swedish child‐

**4.1. Enterovirus in biological samples from patients with type 1 diabetes**

development of T1D (table 3).

*4.1.1. Whole blood*

38 Type 1 Diabetes

the course of T1D.

disease (metabolic decompensation or not)

autoimmunity and/or risk of developing the disease.

The polymerase chain reaction, targeting the 5' non coding region of enteroviral RNA was first used in an English study to detect viral genome in serum taken from 14 children at the onset of T1D and 45 control children matched for age, sex, date of specimen receipt and, as far as possible, geographic area [31]. In this study, a significant greater number of diabetic children had positive PCR results compared with controls (64% vs. 4%). Sequencing of en‐ terovirus PCR products from six positive patients showed a significant homology with cox‐ sackie B3 and B4 viruses, and some common patterns were observed among the sequences from infected diabetic children.

An English team investigated the relationship between enterovirus RNA and T1D in chil‐ dren [108]. One hundred ten children (aged 0-15 years with an average of 7.1 years) with newly diagnosed T1D were recruited. Detection of enterovirus RNA in serum col‐ lected in the week after diagnosis was based on a RT-PCR amplifying the 5' noncod‐ ing region of enterovirus genome. Hundred and eighty-two control children were matched to cases by age (average age: 6.6 years), sex and date of serum collection at the same hospital. The number of newly diagnosed children with a positive RT-PCR was signif‐ icantly higher than in the control group (27% versus 4.9%, p <0.005). Moreover, a sig‐ nificant proportion of diabetic children with a positive RT-PCR were of very young age. Indeed, enteroviruses were detected in 37% (20/54) of T1D children aged under 7 years, whereas only 4.6% (5/111) of corresponding control children were positive for en‐ terovirus RNA (p <0.005). For diabetic children older than 7 years, 17.8% (10/56) were found to be positive for enterovirus RNA sequences, while viral RNA was detected in only 5.6% (4/71) of corresponding controls (p <0.05).

A French study evaluated the possible role of enteroviruses infections in the pathogenesis of T1D (Coutant et al., 2002). Sixteen newly diagnosed T1D children were included in this study. Forty nine control children matched for age, sex, date of venous collection and geo‐ graphic area. A highly sensitive RT-PCR was used to investigate RNA in serum from pa‐ tients and controls. Neutralzation antibodies to coxsackies viruses B1 to B6 were used to characterize the positive PCR samples. Enterovirus RNA was detected by PCR in only 2 of the 16 newly diagnosed T1D children and in only one of the 49 matched controls (p<0.1). Neutralization assay could not detect antibodies against coxsackiesviruses B1 to B6.

Two hundred and six newly diagnosed T1D children and 160 controls were included in an Australian study [37]. Enterovirus-RNA was found in either plasma or stool in 30% (62/206) of newly diagnosed T1D but only in 4 % (6/160) of controls (p<0.001). Case patients, positive for enterovirus RNA had lower C-peptide levels (p=0.04). Case children with enterovirus RNA levels were more likely to have a severe diabetic ketoacidosis (p = 0.03). Enteroviruses were detected in fewer children with HLA haplotype DRB1 \* 03 DQB1 \* 02 (p = 0.02) sug‐ gesting that the likely role of enteroviruses in the development of diabetes is important in some patients with specific genetic risk.

patients and 31% of non diabetic controls. This study showed no difference between diabetic patients and controls regarding the frequency of infection by enterovirus. Whether enterovi‐ ruses acted as non-specific agents with an abnormal immune response of the host, is a ques‐

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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

41

In a Swedish group, Yin and colleagues used RT-PCR to detect enterovirus RNA in PBMC (peripheral blood mononuclear cells) from 24 newly diagnosed children patients [171]. The 24 control children were matched for age, sex and geographical location without evidence of ongoing infection. RT-PCR was performed with primers (groups A and B) corresponding to conserved areas in the 5'non-coding region. With group A primers, 50% (12/24) of newly di‐ agnosed patients had a positive enterovirus RT-PCR, however, control children were nega‐ tive (p<0.001). With group B primers, enterovirus sequences were detected in 46% (11/24) of newly diagnosed patients, and in 29 % (7/24) of control children, but the difference was not statistically significant. Taking into account the results obtained with the two sets of pri‐ mers, the detection of enterovirus RNA was positive in 75% (18/24) of newly diagnosed pa‐

One hundred and twelve diabetic children and 56 healty controls have been included in an Italian study [154]. Enterovirus common capsid antigens were detected by immunofuores‐ cence in a panel of cell lines inoculated with total leucocytes from peripheral blood, and en‐ teroviral RNA was detected in these cultures as well. Enteroviruses were detected by RT-PCR in 93/112 case children (83%) and 4/56 control children (7%), and directly in leukocytes

Enteroviral RNA has been searched in PBMC, plasma, throat and stools of 10 newly diag‐ nosed children and 20 control children [132]. Viral RNA was found in PBMC of 4 patients (40%), in plasma of 2 patients (20%), and in stools in 1 patient, in contrast, no sample was positive in control children. All throat swabs from patients and controls were negative. Ac‐ cording to the authors, a prolonged enteroviral infection could be suspected in these pa‐ tients in front of a positive detection of viral RNA in PBMC and/or plasma together with a

A 10 years old child with a flu-like illness within 3 days prior to admission in hospital for diabetic ketoacidosis died on the 7th day of admission [175]. The autopsy showed infiltra‐ tion of the pancreas islets by lymphocytes with necrosis of beta cells. The inoculation of mouse, monkey and human cell cultures with a homogenate of the patient's pancreas had led to the isolation of a CVB4. Serology showed an 8 fold increase in titer of neutralizing an‐ tibody to this virus between the second hospital day and day of death. Inoculation of mice with this viral isolate led to hyperglycemia, inflammation of the islets of Langerhans and ne‐ crosis of beta cells. Immunoflurescence detected viral antigen in beta cells of mouse pancre‐

A few years later, a British group [52], did not find VP1 by immunohistochemistry in pan‐ creas beta cells of 88 patients who had died at clinical presentation of T1D. In contrast, by

atic section. The virus isolate obtained from this patient is known as CVB4 E2.

at lower frequency. Thirteen cases of familial enterovirus infection were observed.

tion raised by the authors of this study.

tients and only in 29% (7/24) of control children.

negative detection of viral RNA in stool and throat swabs.

*4.1.4. Pancreas*

An Egyptian study included 70 diabetic children who were classified into 2 groups: the first group (I), 40 patients with newly diagnosed diabetic patients (less than one year), the second group (II), 30 children with diabetic patients with more than one year duration of disease [100]. In the control group there were 30 normal healthy children. Enterovirus infection was detected by viral culture of serum samples and confirmation of the results of tissue culture isolation was done by RT-PCR. In addition, anti-CVB IgM and IgG antibodies were searched by enzyme immuno assay. Enterovirus was isolated in group I (47.5%) and group II (23.3%). Neutralization test revealed that most of cases were coxsackievirus B4. In this study, cox‐ sackievirus B IgM antibodies were significantly higher in diabetic patients of group I than those in group II (p<0.01) but there was no significant difference between group I and group II regarding IgG positivity.

A Japanese case-control study encompassed 61 patients with T1D aged 9 months to 40 years and 58 non diabetic subjects aged 1 month to 40 years whose serum was collected the same year [90]. A highly sensitive RT-PCR was used to investigate enterovirus RNA in serum samples. Moreover, neutralizing antibodies against Coxsackievirus and antibodies to GAD were measured and compared with the viral load and the enterovirus genotype. The detec‐ tion of enterovirus was positive in 23 out of 61 patients (37.7%) and in 2 out of 58 controls (3.4%). The positivity of RT-PCR was decreasing by years gradually after the occurrence of T1D, there was neither gender nor age tendency. The sequence analysis of PCR amplicons displayed strong homologies with coxsackievirus B4 in 13 patients out of 23, and the level of neutralizing anti-CVB4 antibodies was significantly high in positive patients in RT-PCR. There was no relationship between the viral load in serum and antibodies against GAD.

A German group searched the enterovirus RNA by RT-PCR in the serum of diabetic chil‐ dren taken soon after the diagnosis of diabetes [104]. Seventeen out of 47 (36%) newly diag‐ nosed diabetic cases were positive for enteroviral RNA whereas 2 out of 50 control subjects were positive (p<0.001).

Cuba is a country with a low incidence of T1D and with a high circulation of enteroviruses. In a Cuban study, the frequency of enteroviral RNA detection by RT-PCR was significantly higher in newly diagnosed T1D children whose diagnosis was made within 10 days before inclusion [26.5% (9/34)] compared to controls [2.9% (2/68)], matched for age, gender, geo‐ graphic origin and date of serum collection (p = 0.0007) [127]. Enterovirus detection was more likely associated with severe diabetic ketoacidosis at onset (pH <7.1, p = 0.03) and high titres of autoantibodies against ICA (p <0.05).

#### *4.1.3. Leucocytes and other biological samples*

An English study included 17 newly diagnosed patients with T1D, 38 previously diagnosed patients with T1D (the median duration of T1D was 4 years) and 43 age and sex matched non-diabetic controls [53]. Enterovirus RNA was detected by PCR in peripheral blood mon‐ onuclear cells in 41 % of newly diagnosed patients with T1D, 39% of previously diagnosed patients and 31% of non diabetic controls. This study showed no difference between diabetic patients and controls regarding the frequency of infection by enterovirus. Whether enterovi‐ ruses acted as non-specific agents with an abnormal immune response of the host, is a ques‐ tion raised by the authors of this study.

In a Swedish group, Yin and colleagues used RT-PCR to detect enterovirus RNA in PBMC (peripheral blood mononuclear cells) from 24 newly diagnosed children patients [171]. The 24 control children were matched for age, sex and geographical location without evidence of ongoing infection. RT-PCR was performed with primers (groups A and B) corresponding to conserved areas in the 5'non-coding region. With group A primers, 50% (12/24) of newly di‐ agnosed patients had a positive enterovirus RT-PCR, however, control children were nega‐ tive (p<0.001). With group B primers, enterovirus sequences were detected in 46% (11/24) of newly diagnosed patients, and in 29 % (7/24) of control children, but the difference was not statistically significant. Taking into account the results obtained with the two sets of pri‐ mers, the detection of enterovirus RNA was positive in 75% (18/24) of newly diagnosed pa‐ tients and only in 29% (7/24) of control children.

One hundred and twelve diabetic children and 56 healty controls have been included in an Italian study [154]. Enterovirus common capsid antigens were detected by immunofuores‐ cence in a panel of cell lines inoculated with total leucocytes from peripheral blood, and en‐ teroviral RNA was detected in these cultures as well. Enteroviruses were detected by RT-PCR in 93/112 case children (83%) and 4/56 control children (7%), and directly in leukocytes at lower frequency. Thirteen cases of familial enterovirus infection were observed.

Enteroviral RNA has been searched in PBMC, plasma, throat and stools of 10 newly diag‐ nosed children and 20 control children [132]. Viral RNA was found in PBMC of 4 patients (40%), in plasma of 2 patients (20%), and in stools in 1 patient, in contrast, no sample was positive in control children. All throat swabs from patients and controls were negative. Ac‐ cording to the authors, a prolonged enteroviral infection could be suspected in these pa‐ tients in front of a positive detection of viral RNA in PBMC and/or plasma together with a negative detection of viral RNA in stool and throat swabs.

#### *4.1.4. Pancreas*

gesting that the likely role of enteroviruses in the development of diabetes is important in

An Egyptian study included 70 diabetic children who were classified into 2 groups: the first group (I), 40 patients with newly diagnosed diabetic patients (less than one year), the second group (II), 30 children with diabetic patients with more than one year duration of disease [100]. In the control group there were 30 normal healthy children. Enterovirus infection was detected by viral culture of serum samples and confirmation of the results of tissue culture isolation was done by RT-PCR. In addition, anti-CVB IgM and IgG antibodies were searched by enzyme immuno assay. Enterovirus was isolated in group I (47.5%) and group II (23.3%). Neutralization test revealed that most of cases were coxsackievirus B4. In this study, cox‐ sackievirus B IgM antibodies were significantly higher in diabetic patients of group I than those in group II (p<0.01) but there was no significant difference between group I and group

A Japanese case-control study encompassed 61 patients with T1D aged 9 months to 40 years and 58 non diabetic subjects aged 1 month to 40 years whose serum was collected the same year [90]. A highly sensitive RT-PCR was used to investigate enterovirus RNA in serum samples. Moreover, neutralizing antibodies against Coxsackievirus and antibodies to GAD were measured and compared with the viral load and the enterovirus genotype. The detec‐ tion of enterovirus was positive in 23 out of 61 patients (37.7%) and in 2 out of 58 controls (3.4%). The positivity of RT-PCR was decreasing by years gradually after the occurrence of T1D, there was neither gender nor age tendency. The sequence analysis of PCR amplicons displayed strong homologies with coxsackievirus B4 in 13 patients out of 23, and the level of neutralizing anti-CVB4 antibodies was significantly high in positive patients in RT-PCR. There was no relationship between the viral load in serum and antibodies against GAD.

A German group searched the enterovirus RNA by RT-PCR in the serum of diabetic chil‐ dren taken soon after the diagnosis of diabetes [104]. Seventeen out of 47 (36%) newly diag‐ nosed diabetic cases were positive for enteroviral RNA whereas 2 out of 50 control subjects

Cuba is a country with a low incidence of T1D and with a high circulation of enteroviruses. In a Cuban study, the frequency of enteroviral RNA detection by RT-PCR was significantly higher in newly diagnosed T1D children whose diagnosis was made within 10 days before inclusion [26.5% (9/34)] compared to controls [2.9% (2/68)], matched for age, gender, geo‐ graphic origin and date of serum collection (p = 0.0007) [127]. Enterovirus detection was more likely associated with severe diabetic ketoacidosis at onset (pH <7.1, p = 0.03) and high

An English study included 17 newly diagnosed patients with T1D, 38 previously diagnosed patients with T1D (the median duration of T1D was 4 years) and 43 age and sex matched non-diabetic controls [53]. Enterovirus RNA was detected by PCR in peripheral blood mon‐ onuclear cells in 41 % of newly diagnosed patients with T1D, 39% of previously diagnosed

some patients with specific genetic risk.

40 Type 1 Diabetes

II regarding IgG positivity.

were positive (p<0.001).

titres of autoantibodies against ICA (p <0.05).

*4.1.3. Leucocytes and other biological samples*

A 10 years old child with a flu-like illness within 3 days prior to admission in hospital for diabetic ketoacidosis died on the 7th day of admission [175]. The autopsy showed infiltra‐ tion of the pancreas islets by lymphocytes with necrosis of beta cells. The inoculation of mouse, monkey and human cell cultures with a homogenate of the patient's pancreas had led to the isolation of a CVB4. Serology showed an 8 fold increase in titer of neutralizing an‐ tibody to this virus between the second hospital day and day of death. Inoculation of mice with this viral isolate led to hyperglycemia, inflammation of the islets of Langerhans and ne‐ crosis of beta cells. Immunoflurescence detected viral antigen in beta cells of mouse pancre‐ atic section. The virus isolate obtained from this patient is known as CVB4 E2.

A few years later, a British group [52], did not find VP1 by immunohistochemistry in pan‐ creas beta cells of 88 patients who had died at clinical presentation of T1D. In contrast, by using the same method VP1 protein was found in cardiac myocytes from 12 of the 20 pa‐ tients whose cause of death was an acute coxsackievirus B myocarditis, and in seven of these positive cases, insulitis was observed and VP1 was detected in islet endocrine cells, but rare‐ ly in exocrine pancreas. Together, these data suggested that the beta cell destruction in pa‐ tients with fatal diabetes was unlikely related to a direct cytopathic effect of coxsackievirus B, however the role of viruses in the destruction of beta cells through an autoimmune mech‐ anism can not be excluded.

The prevalence of enteroviral capsid protein (VP1) in pancreatic autopsy tissue from 72 newly diagnosed T1D children and a large cohort of controls has been studied by immuno‐ histochemical staining by a british group [122]. The cell subtypes infected with enteroviruses were identified by immunofluorescence. The criterion of positivity was the presence of at least one intensely stained endocrine cell in an islet within any given section. According to this criterion, 61% (44/72) of diabetic children were positive in immunohistochemistry ver‐ sus 7.7% (3/39) of control children (p <0.001). There was no significant difference regarding age or gender between the VP1-positive and VP1-negative groups however the duration of diabetes seemed to be lower in the VP1-positive group (2.32 months vs 16.5 months; p=0.06).

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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

43

Entroviruses are present in stools of infected individuals [125]. The hypothesis of the role of enteroviruses in T1D prompted researchers to look for these viruses in stools of patients.

An Italian group investigated enteroviruses in stools from 43 newly diagnosed diabetic chil‐ dren and 22 control children [42]. Stools and serum samples were collected within 2 months from the beginning of diabetes symptoms. In order to isolate enterovirus, stools were inoculat‐ ed to cell cultures and in suckling mice. Neutralizing antibodies to coxsackie virus B4 and anticoxsackie viruses B1 to B6 complement fixing antibodies were measured. There was one case with high antibodies against coxsackie B4 virus but no enterovirus was isolated from stools. A 16 month-old child with a predisposing HLA group (B18 DRw3) developed diabetes [21]. The disease outcame at hospital on the third day of steroid therapy for febrile purpura with‐ in the week of diphtheria/pertussis/tetanus and polio vaccination. Coxsackievirus B5 was isolated from stools and serologic studies showed a rise in titer of neutralizing antibodies directed to that isolate from less than 10 on the first day to 640 on the eleventh day. The sud‐ den onset of T1D in the course an acute coxsackievirus B5 infection suggests the potential

Different virological methods were used in a Finnish study to evaluate whether enterovirus‐ es can be found in small intestinal mucosa of 12 patients with T1D (age: 18 to 53, 2 out of them were male) and 10 non-diabetic subjects (age 23 to 71, 3 out of them were male) [114]. These individuals underwent gastroscopy for gastrointestinal symptoms and intestinal mu‐ cosa biopsies were taken for morphological analysis, which did not reveal any abnormality. To analyse the presence of enteroviruses in intestinal biopsy samples, immunohistochemis‐ try was used for detecting the viral protein VP1, and in situ hybridization. RT-PCR were used for detecting viral RNA. Six out of 12 (50%) diabetic patients were positive for entero‐ viral RNA by in situ hybridization, whereas all control subjects were negative (p = 0.015). Two of these positive patients had enteroviral RNA in the cells of lamina propria; four were positive in the epithelial cells of villi, in the crypts and in the cells of lamina propria. Immu‐ nohistochemistry was positive in 9 out of 12 (75%) of diabetic patients but only in 10% (1/10) of control subjects (p = 0.004): the protein VP1 was mainly localized in the epithelium. Viral RNA was found by RT-PCR in a frozen sample from one of the 4 diabetic patients who were

involvement of this virus in the disease in that case.

*4.1.5. Stools*

*4.1.6. Intestine*

A few years later, another group investigated the presence of enteroviral RNA in the pan‐ creas of 2 children patients with fatal acute-onset T1D and 5 controls by using RT-PCR and Southern blot hybridization [17]. The detection of Enteroviral RNA, and other viral genome (cytomegalovirus, mumps and rubella) was negative in every case.

The relationship between enterovirus and T1D and the type of pancreatic cells infected with enteroviruses has been invesigated by a finish group [172]. The study included 12 newborn infants who died of fulminant infection with enteroviruses (myocarditis in most cases). Au‐ topsy pancreases from 65 patients with T1D and 40 control subjects matched for age and sex were also studied for presence of enteroviral RNA by in situ hybridisation. Enteroviral RNA was detected in pancreas of 58% (7/12) of the 12 newborns; the enterovirus-positive cells were detected in numerous pancreatic islets and in some duct cells but not in exocrine pan‐ creas. In situ hybridisation identified enteroviruses in 6% (4/65) of diabetic patients. Entero‐ viral RNA was located exclusively in islets. None of the control subjects was positive for enteroviral RNA.

More recently, an Italian team studied the relationship between enterovirus infection, in‐ flammation of pancreatic beta cells, autoimmunity and beta cell function [43]. Six newly di‐ agnosed T1D patients (1 week to 9 months) and 26 control organ donors were included in this study. Immunohistochemistry, electron microscopy, RT-PCR and sequencing, and virus isolation in cell culture were used to detect enteroviruses in pancreatic autopsic tissue. En‐ teroviral RNA was detected in 3 out of 6 diabetic patients but not in controls. Infection was specific of beta cells with non-destructive insulitis and with naturel killer cell infiltration. There was not apparent reduction of islet beta cells in these patients. The virus isolated from one of these 3 patients, identified as CVB4 was able to infect human pancreatic beta cells of nondiabetic multiorgan donors. Viral inclusions and signs of pyknosis were observed by electronic microscopy, and a loss of beta cell function was assessed by insulin secretion re‐ sponse to glucose, arginine and glibenclamide. These data show that enterovirus could in‐ fect beta cells in patients with T1D and that these viruses could be responsible for inflammation and functional abnormalities of these cells.

Recently, authors raised the issue of the relevance of pancreas tissue samples to display the relationship between enterovirus infection and type 1diabetes, since no enteroviral RNA was detected by RT-PCR in samples from pancreatic organ donors with diabetes [158]. Fur‐ ther investigation with pancreas from additionnal donors are needed to address the issue of the persistence of enteroviruses in this organ. Whether enteroviruses are present in pancreas tissue at the time of symptom onset should be investigated but tissue samples can not be easily obtained by biopsy in the case of this organ.

The prevalence of enteroviral capsid protein (VP1) in pancreatic autopsy tissue from 72 newly diagnosed T1D children and a large cohort of controls has been studied by immuno‐ histochemical staining by a british group [122]. The cell subtypes infected with enteroviruses were identified by immunofluorescence. The criterion of positivity was the presence of at least one intensely stained endocrine cell in an islet within any given section. According to this criterion, 61% (44/72) of diabetic children were positive in immunohistochemistry ver‐ sus 7.7% (3/39) of control children (p <0.001). There was no significant difference regarding age or gender between the VP1-positive and VP1-negative groups however the duration of diabetes seemed to be lower in the VP1-positive group (2.32 months vs 16.5 months; p=0.06).

### *4.1.5. Stools*

using the same method VP1 protein was found in cardiac myocytes from 12 of the 20 pa‐ tients whose cause of death was an acute coxsackievirus B myocarditis, and in seven of these positive cases, insulitis was observed and VP1 was detected in islet endocrine cells, but rare‐ ly in exocrine pancreas. Together, these data suggested that the beta cell destruction in pa‐ tients with fatal diabetes was unlikely related to a direct cytopathic effect of coxsackievirus B, however the role of viruses in the destruction of beta cells through an autoimmune mech‐

A few years later, another group investigated the presence of enteroviral RNA in the pan‐ creas of 2 children patients with fatal acute-onset T1D and 5 controls by using RT-PCR and Southern blot hybridization [17]. The detection of Enteroviral RNA, and other viral genome

The relationship between enterovirus and T1D and the type of pancreatic cells infected with enteroviruses has been invesigated by a finish group [172]. The study included 12 newborn infants who died of fulminant infection with enteroviruses (myocarditis in most cases). Au‐ topsy pancreases from 65 patients with T1D and 40 control subjects matched for age and sex were also studied for presence of enteroviral RNA by in situ hybridisation. Enteroviral RNA was detected in pancreas of 58% (7/12) of the 12 newborns; the enterovirus-positive cells were detected in numerous pancreatic islets and in some duct cells but not in exocrine pan‐ creas. In situ hybridisation identified enteroviruses in 6% (4/65) of diabetic patients. Entero‐ viral RNA was located exclusively in islets. None of the control subjects was positive for

More recently, an Italian team studied the relationship between enterovirus infection, in‐ flammation of pancreatic beta cells, autoimmunity and beta cell function [43]. Six newly di‐ agnosed T1D patients (1 week to 9 months) and 26 control organ donors were included in this study. Immunohistochemistry, electron microscopy, RT-PCR and sequencing, and virus isolation in cell culture were used to detect enteroviruses in pancreatic autopsic tissue. En‐ teroviral RNA was detected in 3 out of 6 diabetic patients but not in controls. Infection was specific of beta cells with non-destructive insulitis and with naturel killer cell infiltration. There was not apparent reduction of islet beta cells in these patients. The virus isolated from one of these 3 patients, identified as CVB4 was able to infect human pancreatic beta cells of nondiabetic multiorgan donors. Viral inclusions and signs of pyknosis were observed by electronic microscopy, and a loss of beta cell function was assessed by insulin secretion re‐ sponse to glucose, arginine and glibenclamide. These data show that enterovirus could in‐ fect beta cells in patients with T1D and that these viruses could be responsible for

Recently, authors raised the issue of the relevance of pancreas tissue samples to display the relationship between enterovirus infection and type 1diabetes, since no enteroviral RNA was detected by RT-PCR in samples from pancreatic organ donors with diabetes [158]. Fur‐ ther investigation with pancreas from additionnal donors are needed to address the issue of the persistence of enteroviruses in this organ. Whether enteroviruses are present in pancreas tissue at the time of symptom onset should be investigated but tissue samples can not be

(cytomegalovirus, mumps and rubella) was negative in every case.

inflammation and functional abnormalities of these cells.

easily obtained by biopsy in the case of this organ.

anism can not be excluded.

42 Type 1 Diabetes

enteroviral RNA.

Entroviruses are present in stools of infected individuals [125]. The hypothesis of the role of enteroviruses in T1D prompted researchers to look for these viruses in stools of patients.

An Italian group investigated enteroviruses in stools from 43 newly diagnosed diabetic chil‐ dren and 22 control children [42]. Stools and serum samples were collected within 2 months from the beginning of diabetes symptoms. In order to isolate enterovirus, stools were inoculat‐ ed to cell cultures and in suckling mice. Neutralizing antibodies to coxsackie virus B4 and anticoxsackie viruses B1 to B6 complement fixing antibodies were measured. There was one case with high antibodies against coxsackie B4 virus but no enterovirus was isolated from stools.

A 16 month-old child with a predisposing HLA group (B18 DRw3) developed diabetes [21]. The disease outcame at hospital on the third day of steroid therapy for febrile purpura with‐ in the week of diphtheria/pertussis/tetanus and polio vaccination. Coxsackievirus B5 was isolated from stools and serologic studies showed a rise in titer of neutralizing antibodies directed to that isolate from less than 10 on the first day to 640 on the eleventh day. The sud‐ den onset of T1D in the course an acute coxsackievirus B5 infection suggests the potential involvement of this virus in the disease in that case.

#### *4.1.6. Intestine*

Different virological methods were used in a Finnish study to evaluate whether enterovirus‐ es can be found in small intestinal mucosa of 12 patients with T1D (age: 18 to 53, 2 out of them were male) and 10 non-diabetic subjects (age 23 to 71, 3 out of them were male) [114]. These individuals underwent gastroscopy for gastrointestinal symptoms and intestinal mu‐ cosa biopsies were taken for morphological analysis, which did not reveal any abnormality. To analyse the presence of enteroviruses in intestinal biopsy samples, immunohistochemis‐ try was used for detecting the viral protein VP1, and in situ hybridization. RT-PCR were used for detecting viral RNA. Six out of 12 (50%) diabetic patients were positive for entero‐ viral RNA by in situ hybridization, whereas all control subjects were negative (p = 0.015). Two of these positive patients had enteroviral RNA in the cells of lamina propria; four were positive in the epithelial cells of villi, in the crypts and in the cells of lamina propria. Immu‐ nohistochemistry was positive in 9 out of 12 (75%) of diabetic patients but only in 10% (1/10) of control subjects (p = 0.004): the protein VP1 was mainly localized in the epithelium. Viral RNA was found by RT-PCR in a frozen sample from one of the 4 diabetic patients who were positive in both in situ hybridization and immunohistochemistry. There was no relationship between the detection of enteroviral RNA in gut mucosa of diabetic patients and duration of diabetes, gender, HLA type or hyperglycemia.

sion to T1D following detection of enteroviral RNA in serum, in a 4-month interval, was sig‐ nificantly increased compared with negative detection. In contrast, the presence of enteroviral RNA in rectal swabs did not predict progression to T1D, which is in agreement with the results of the MIDIA study including 911 Norwegian children identified at birth

Thirty height children with an increased genetic susceptibility to diabetes followed-up from birth who have progressed to T1D and 140 control children matched for sexe, date of birth, hospital district and HLA-DQ-conferred genetic susceptibility to T1D were included in the finnish type 1 Diabetes Prediction and Prevention study (DiPP) [115]. Serum samples were analysed for enterovirus RNA by RT-PCR: 5.1% of samples were enterovirus RNA positive in case children but only 1, 9% in control children (p<0. 01). In boys, the detection of entero‐ virus RNA during the 6 months preceding the discovery of autoantibodies was associated

> **Methods of detection**

**Reference country**

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45

RT-PCR Andréoletti et al., 1997 France

RT-PCR Chehadeh et al., 2000 France

RT-PCR Dahlquist et al., 2004 Sweden

RT-PCR Clements et al., 1995 England

RT-PCR Nairn et al., 1999 England

RT-PCR Coutant et al., 2002 France

Cell culture Maha et al., 2003 Egypt

RT-PCR Kawashima et al., 2004 Japan

RT-PCR Moya-Suri et al., 2005 Germany

RT-PCR Sarmiento et al., 2007 Cuba

RT-PCR Craig et al., 2003 Australia

with a HLA genotype conferring a risk of T1D [149].

**Children/ adults patients**

**Positives cases/ Controls p value**

p <0.01

p \*

p\*

p=0.04

p<0.005

p>0.05

p <0.05

p<0.05

p<0.001

p=0.0007

plasma or stools p<0.001

with a risk of diabetes (p<0.01).

**Number of Cases/ Controls**

Whole blood 24/27 0/24 6/0

Whole blood 56/37 25/31 21/0

Woole Blood 600/600 600/0 27/14

Serum 14/45 14/0 9/2

Serum 110/182 110/0 30/9

Serum 16/49 16/0 2/1

Serum 70/30 70/0 26/0

Serum 61/58 NI 23/2

Serum 47/50 47/0 17/2

Serum 34/68 34/0 9/2

206/160 206/0 62/6

Plasma stools

**Biological samples**

The discrepancy in results obtained by RT-PCR and in situ hybridization could be explained by the fact that intestinal biopsy samples were obtained from two sites of the intestine, and by differences in samples preparation. These results display that subjects with T1D have en‐ teroviral components in their gut mucosa.

#### **4.2. Enterovirus in biological samples from individuals at high-risk of diabetes**

A Finnish prospective study concerned children with prediabetic state, which were derived from a previous study "Childhood Diabetes in Finland" (DiMe) [99]. The study investigated enterovirus RNA in 93 serum samples from 11 prediabetic children who progressed to T1D during the follow-up. One hundred and eight serum samples from 34 control children who participated in the same cohort but did not develop autoimmunity against beta cells or T1D were examined. In this study, serum samples from 47 children with newly diagnosed T1D were also analysed. Antibodies against islet cells (ICA), glutamic acid decarboxylase (GA‐ DA), insulin (IAA) and the protein tyrosine phosphatase-related IA-2 protein (IA2-A) were analysed. Antibodies against coxsackie viruses B1 to B6 were measured by neutralization as‐ say. Enterovirus RNA was found in 12 %( 11/93) of follow-up samples from prediabetic chil‐ dren compared to only 2% (2/108) of follow-up samples from matched controls (p<0.01). Viral RNA was detected in none (0/47) of the serum samples obtained from diabetic chil‐ dren. The presence of enteroviral RNA was associated with a concomitant increase in ICA (p <0.01) and GADA (p <0.05), whereas no increase was observed in the rates of IAA and IA-2A. This study suggests that enterovirus genome can be found in serum of individuals and that it is associated with the induction of autoimmunity several years before the onset of symptoms. The presence of enterovirus RNA in serum of prediabetic children has been studied in Cuba [127]. This study encompassed 32 children positive for antibodies against ICA having a first-degree relative with T1D, 31 children, negative for antibodies against ICA having a diabetic first-degree relative, and 194 controls, who were matched for age, gender, geographic origin and date of serum collection. Enterovirus RNA was found in 15.6% (5/32) of islet autoantibody-positive first-degree relatives children, whereas all controls were nega‐ tive for enteroviral genome (P = 0.003). Enterovirus RNA was found in 3.2% of 31 children, negative for antibodies against ICA having a diabetic first-degree relative, and in 1.6% of controls.

After seroconversion for islet antibodies (against GAD, insulin, IA-2), serum and rectal swabs were collected every 3-6 months until diagnosis of diabetes in the Diabetes and Auto‐ immunity Study in the Young (DAISY) encompassing 2,365 american genetically predis‐ posed children for islet autoimmunity and T1D, according to HLA, and siblings or offspring of people with T1D (regardless of their genotype) [141]. Fifty of the 140 children who sero‐ converted to positivity for islet autoantibodies progressed to T1D. The prevalence of entero‐ viral RNA in serum and rectal swabs as displayed by RT-PCR declined with age and seemed to be higher at visits positive for multiple islet autoantibodies. The risk of progres‐ sion to T1D following detection of enteroviral RNA in serum, in a 4-month interval, was sig‐ nificantly increased compared with negative detection. In contrast, the presence of enteroviral RNA in rectal swabs did not predict progression to T1D, which is in agreement with the results of the MIDIA study including 911 Norwegian children identified at birth with a HLA genotype conferring a risk of T1D [149].

positive in both in situ hybridization and immunohistochemistry. There was no relationship between the detection of enteroviral RNA in gut mucosa of diabetic patients and duration of

The discrepancy in results obtained by RT-PCR and in situ hybridization could be explained by the fact that intestinal biopsy samples were obtained from two sites of the intestine, and by differences in samples preparation. These results display that subjects with T1D have en‐

A Finnish prospective study concerned children with prediabetic state, which were derived from a previous study "Childhood Diabetes in Finland" (DiMe) [99]. The study investigated enterovirus RNA in 93 serum samples from 11 prediabetic children who progressed to T1D during the follow-up. One hundred and eight serum samples from 34 control children who participated in the same cohort but did not develop autoimmunity against beta cells or T1D were examined. In this study, serum samples from 47 children with newly diagnosed T1D were also analysed. Antibodies against islet cells (ICA), glutamic acid decarboxylase (GA‐ DA), insulin (IAA) and the protein tyrosine phosphatase-related IA-2 protein (IA2-A) were analysed. Antibodies against coxsackie viruses B1 to B6 were measured by neutralization as‐ say. Enterovirus RNA was found in 12 %( 11/93) of follow-up samples from prediabetic chil‐ dren compared to only 2% (2/108) of follow-up samples from matched controls (p<0.01). Viral RNA was detected in none (0/47) of the serum samples obtained from diabetic chil‐ dren. The presence of enteroviral RNA was associated with a concomitant increase in ICA (p <0.01) and GADA (p <0.05), whereas no increase was observed in the rates of IAA and IA-2A. This study suggests that enterovirus genome can be found in serum of individuals and that it is associated with the induction of autoimmunity several years before the onset of symptoms. The presence of enterovirus RNA in serum of prediabetic children has been studied in Cuba [127]. This study encompassed 32 children positive for antibodies against ICA having a first-degree relative with T1D, 31 children, negative for antibodies against ICA having a diabetic first-degree relative, and 194 controls, who were matched for age, gender, geographic origin and date of serum collection. Enterovirus RNA was found in 15.6% (5/32) of islet autoantibody-positive first-degree relatives children, whereas all controls were nega‐ tive for enteroviral genome (P = 0.003). Enterovirus RNA was found in 3.2% of 31 children, negative for antibodies against ICA having a diabetic first-degree relative, and in 1.6% of

After seroconversion for islet antibodies (against GAD, insulin, IA-2), serum and rectal swabs were collected every 3-6 months until diagnosis of diabetes in the Diabetes and Auto‐ immunity Study in the Young (DAISY) encompassing 2,365 american genetically predis‐ posed children for islet autoimmunity and T1D, according to HLA, and siblings or offspring of people with T1D (regardless of their genotype) [141]. Fifty of the 140 children who sero‐ converted to positivity for islet autoantibodies progressed to T1D. The prevalence of entero‐ viral RNA in serum and rectal swabs as displayed by RT-PCR declined with age and seemed to be higher at visits positive for multiple islet autoantibodies. The risk of progres‐

**4.2. Enterovirus in biological samples from individuals at high-risk of diabetes**

diabetes, gender, HLA type or hyperglycemia.

44 Type 1 Diabetes

teroviral components in their gut mucosa.

controls.

Thirty height children with an increased genetic susceptibility to diabetes followed-up from birth who have progressed to T1D and 140 control children matched for sexe, date of birth, hospital district and HLA-DQ-conferred genetic susceptibility to T1D were included in the finnish type 1 Diabetes Prediction and Prevention study (DiPP) [115]. Serum samples were analysed for enterovirus RNA by RT-PCR: 5.1% of samples were enterovirus RNA positive in case children but only 1, 9% in control children (p<0. 01). In boys, the detection of entero‐ virus RNA during the 6 months preceding the discovery of autoantibodies was associated with a risk of diabetes (p<0.01).



**5.1. In vivo studies in animal models**

with coxsackievirus B (CVB) (figure 3).

*5.1.1. Enterovirus and immune system*

infection and pancreatitis [86].

are still poorly understood.

In order to analyse the hypothesis that enterovirus infections enhance or elicit autoimmune disorders such as T1D, a significant body of evidence is derived from investigations using animal models. Most of them used to explore research hypotheses regarding the relation‐ ship between enteroviruses and type 1 diabetes are mouse models (NOD, C57BL/k, C57BL/6, SJL/J, DBA/2, SWR/J, BALB/c, B10, CD-1…) [83]. Despite their limitation in diseas‐ es investigations, experimental models have greatly contributed to our knowledge of human diseases. The predominance of murine models for the investigation of the relationship be‐ tween enteroviruses and T1D is due, among others, to a physiology relatively similar to that of human beings and the presence of specific receptors, the more prominent of them could be the coxsackievirus and adenovirus receptor (CAR) which is a receptor for coxsackievirus B [86]. Therefore experimental datas have been obtained from models based on infection

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Experimental in vivo studies have contributed improving our understanding of genetic and immunological implications, enteroviral tropism and mechanisms of pancreatic β-cells de‐ struction in the context of enteroviral infection [83]. Enteroviruses generally infect the exo‐ crine pancreas, but some strains preferentially infect islets. Some studies have addressed the role of CAR, the main receptor for CVB entry into host cell, in enteroviral tropism and target organ infection. CAR is expressed by intestine, pancreas and heart epithelial cells, as well as cardiomyocytes [54]. In transgenic mice CVB3 titers were markedly reduced in CAR-defi‐ cient tissues and pancreatic CAR deletion induced a strong attenuation of pancreatic CVB3

The development of innate and adaptive immune responses is mediated by type I interfer‐ ons (IFNs) produced early during viral infection to induce an antiviral state within target cells. Experimental studies have shown that mice deficient in type I IFNs receptor are more susceptible and die more rapidly than controls when infected with CVB3 [169, 40]. An effi‐ cient immune response depends on rapid recognition of viruses by the innate immune sys‐ tem and this recognition is primarily achieved by pattern-recognition receptors such as tolllike receptors (TLRs), retinoid-inducible gene 1-like receptors (RIG-1) and NOD-like receptors. It is noteworthy that interactions between NOD-like receptors and enteroviruses

Toll-like receptors are transmembrane glycoproteins expressed on the cytoplasmic mem‐ brane or in intracellular vesicals of several immune and non-immune cell populations; while RIG-I-like receptors, represented by RIG-I and the interferon-induced with helicase C do‐ main 1 (IFIH-1), also called melanoma differentiation-associated gene 5 (MDA5) are ex‐ pressed in the cytosol of most cell types [91]. Among TLRs, TLR3, known to be doublestranded RNA sensor on monocytes, is known to be crucial for the survival of mice infected with CVB4 [123]; and the production of cytokines by murine plasmocytoid dendritic cells have been shown to be closely linked with CVB detection and recognition by TLR7 [168]. The MDA5 is in turn essential for type I IFNs responses to CVB, since MDA5 knockout mice

**Table 3.** Detection of enterovirus and/or their components (RNA, proteins) in biological samples of patients with type 1 diabetes. PBMC: Peripheral Blood Mononuclear Cells, RT-PCR: Retrotranscription Polymerase Chain Reaction, IHC: Immunohistochemistry, HIS: Hybridization in situ, NI: Not Indicated, p\*: p value not mentioned.
