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In type 1 diabetes, pancreatic beta cells are destroyed by chronic autoimmune responses. The disease develops in genetically susceptible individuals, but a role for environmental factors has been postulated. Viral infections have long been considered as candidates for environmental triggers but, given the lack of evidence for an acute, widespread, cytopathic effect in the pancreas in type 1 diabetes or for a closely related temporal association of diabetes onset with such infections, a role for viruses in type 1 diabetes remains unproven. Moreover, viruses have rarely been isolated from the pancreas of individuals with type 1 diabetes, mainly (but not solely) due to the inaccessibility of the organ. Here, we review past and recent literature to evaluate the proposals that chronic, recurrent and, possibly, persistent enteroviral infections occur in pancreatic beta cells in type 1 diabetes. We also explore whether these infections may be sustained by different virus strains over time and whether multiple viral hits can occur during the natural history of type 1 diabetes. We emphasise that only a minority of beta cells appear to be infected at any given time and that enteroviruses may become replication defective, which could explain why they have been isolated from the pancreas only rarely. We argue that enteroviral infection of beta cells largely depends on the host innate and adaptive immune responses, including innate responses mounted by beta cells. Thus, we propose that viruses could play a role in type 1 diabetes on multiple levels, including in the triggering and chronic stimulation of autoimmunity and in the generation of inflammation and the promotion of beta cell dysfunction and stress, each of which might then contribute to autoimmunity, as part of a vicious circle. We conclude that studies into the effects of vaccinations and/or antiviral drugs (some of which are currently on-going) is the only means by which the role of viruses in type 1 diabetes can be finally proven or disproven.

Aims/hypothesis: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes., Methods: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue., Results: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1., Conclusions/interpretation: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

Aims/hypothesis: Enterovirus (e.g. Coxsackie B virus serotypes [CVBs]) infections may be associated with development of type 1 diabetes. Studies conducted in several European countries have, however, shown an inverse correlation between the incidence of type 1 diabetes and the prevalence of enterovirus infections. These findings could in part be explained by an extension of the poliovirus hypothesis, suggesting that the absence of maternally transferred antibodies protecting offspring from early infection increases the risk for diabetes development. Experimental evidence supporting this hypothesis in type 1 diabetes is, however, lacking. As maternally transferred protection from infection is a crucial component of the extended poliovirus hypothesis, we here tested the hypothesis that previously infected females transfer protection against infection and diabetes to offspring., Methods: The induction of CVB-specific maternal antibodies and transfer of protection from virus infection, replication and development of virus-induced diabetes to offspring was assessed using NOD and Socs1-transgenic NOD mice., Results: Infected mice produced neutralising antibodies to CVB. Offspring from infected females were positive for neutralising antibodies and were strongly protected from both infection and experimental diabetes., Conclusions/interpretation: Our study shows that maternally transferred antibodies protect offspring from enterovirus infection and virus-induced diabetes. This suggests that the absence of maternally provided protection increases the risk for severe outcomes after an enterovirus infection in offspring. Moreover, our findings may have implications for the design of prospective studies aimed at investigating the possible role of enterovirus infections in the aetiology of human type 1 diabetes.

Aims/hypothesis: We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study., Methods: Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected., Results: IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype., Conclusions/interpretation: Our data show that children who seroconverted for diabetes-associated auto-antibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes.

Aims/hypothesis: High frequencies of T-cell receptor (TCR) V beta 7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR V beta gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients., Methods: T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes., Results: T-cell receptor V beta 7 gene family values were higher in recently-diagnosed diabetic patients (10.5% +/- 3.7) than in age-matched non-diabetic control subjects (5.1% +/- 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i.e., converters), T-cell receptor V beta 7 gene expression showed values consistently above 10% (p < 0.0005). Long-standing diabetic patients showed lower percentage of V beta 7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of V beta 7 gene transcripts., Conclusion/interpretation: The deviation in the T-cell receptor V beta repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes.

A mild diabetes was induced in mice following the inoculation of a myocardial variant of encephalomyocarditis virus. The majority of infected mice developed a transient hyperglycaemia and only a few mice exhibited a chronic elevation of blood glucose. Infected mice were selected for study if their non-fasting blood glucose levels were above two standard deviations of values in control mice for at least 5 consecutive days. The pancreas from selected inoculated mice and control mice was removed 60 days after infection; random blood glucose levels at this time had returned to normal. Isolated islets of Langerhans from such previously 'diabetic' mice were used to determine the rate of insulin secretion, insulin biosynthesis and cyclic AMP accumulation in response to various glucose concentrations. At glucose (16 mmol/l), both insulin release and biosynthesis were depressed in previously infected animals. At glucose (2 and 10 mmol/l), while insulin biosynthesis was unchanged in infected animals, insulin release was increased. Cyclic AMP accumulation in response to glucose (20 mmol/l) was found to be significantly elevated in islets from infected animals and especially in response to glucose (20 mmol/l) in the presence of isobutyl methyl xanthine (1 mmol/l). Infected mice exhibited a reduction of total pancreatic insulin content in comparison with control mice. The insulin content of isolated islets of Langerhans from infected and control mice was found to be the same. Serum insulin levels, however, from infected mice were higher than in control sera.(ABSTRACT TRUNCATED AT 250 WORDS)

The eyes, kidneys and pancreas of mice (SJL/J) infected with encephalomyocarditis virus were examined by light and electron microscopy. Diabetic mice with the longest duration (6 months) of diabetes showed marked renal and ocular alterations. Fasting blood glucose levels were 17.8-21.9 mmol/l and glycosuria was present. Clinically, based on ophthalmoscopy and fluorescein angiography, retinal vessels were normal. Histologically, moderately decreased numbers of pericytes were noted following trypsin digestion. The basement membrane of inner retinal vessels showed significant thickening in diabetic mice. Corneal epithelial oedema was present and surface microvillus projections were decreased compared with control mice. The kidneys of the same animals showed nodular and diffuse glomerulosclerosis and mesangial thickening. Electron microscopy showed excessive accumulation of basement membrane-like material in the mesangium and the peripheral glomerular region. Histologically, moderate to advanced kidney disease was associated with relatively early retinopathy.

The D variant of encephalomyocarditis virus (EMCV-D) induces a diabetes mellitus-like disease in male SJL/J mice. Other inbred strains, while resistant to the diabetogenic effect, exhibit strikingly different responses to this virus. In these studies, infection of diabetes resistant C3H mice with the D variant produces massive acute pancreatitis with little apparent direct islet cell involvement. This exocrine tropism is not altered when C3H mice with an inherent macrophage defect are infected, and appears to be a gender-specific phenomenon, with female C3H mice resistant to this exocrine involvement. Long-term infection of both male and female C3H mice does not change their response to the virus. Castration of male C3H mice, using a protocol that has been reported to block the diabetogenic effect of this virus, does not alter the development of this acinar lesion. The B variant of EMCV does not induce acinar destruction, nor is it diabetogenic. However, preinfection with the B variant 3 days prior to infection with the D variant does protect against the development of the exocrine lesion. Coinfection with equal doses of the two variants also protects against this lesion, as does coinfection with a lower dose of B variant. Therefore, the host response that is generated against the B variant appears to be responsible for this protection from D variant exocrine destruction. Due to the short time frame, it is unlikely that this protection is the result of an antibody response. Rather, this data is more consistent with an interferon response generated against the B variant that would inhibit replication of the D variant.