**4. Relationship between enteroviruses and autoimmune type 1 diabetes: clinical studies**

Enterovirus infections are among the main environmental risk factors for autoimmune T1D and they have been diagnosed more frequently in T1D patients than in healthy subjects. In this section we report the different studies conducted to investigate the relationship between 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 development of T1D (table 3).

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%)

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

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

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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

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

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).

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‐

Neutralization assay could not detect antibodies against coxsackiesviruses B1 to B6.

control children (p=0.04).

*4.1.2. Serum and plasma*

from infected diabetic children.

only 5.6% (4/71) of corresponding controls (p <0.05).

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 autoimmunity and/or risk of developing the disease.

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