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The rapidly increasing incidence of type 1 diabetes implies that environmental factors are involved in the pathogenesis. Enteroviruses are among the suspected environmental triggers of the disease, and the interest in exploring the possibilities to develop vaccines against these viruses has increased. Our objective was to identify enterovirus serotypes that could be involved in the initiation of the disease process by screening neutralizing antibodies against 41 different enterovirus types in a unique longitudinal sample series from a large prospective birth-cohort study. The study participants comprised 183 case children testing persistently positive for at least two diabetes-predictive autoantibodies and 366 autoantibody-negative matched control children. Coxsackievirus B1 was associated with an increased risk of β-cell autoimmunity. This risk was strongest when infection occurred a few months before autoantibodies appeared and was attenuated by the presence of maternal antibodies against the virus. Two other coxsackieviruses, B3 and B6, were associated with a reduced risk, with an interaction pattern, suggesting immunological cross-protection against coxsackievirus B1. These results support previous observations suggesting that the group B coxsackieviruses are associated with the risk of type 1 diabetes. The clustering of the risk and protective viruses to this narrow phylogenetic lineage supports the biological plausibility of this phenomenon.

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2-targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months' observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Objective: To investigate whether enterovirus infections predict progression to type 1 diabetes in genetically predisposed children repeatedly positive for islet autoantibodies., Research Design and Methods: Since 1993, the Diabetes and Autoimmunity Study in the Young (DAISY) has followed 2,365 genetically predisposed children for islet autoimmunity and type 1 diabetes. Venous blood and rectal swabs were collected every 3-6 months after seroconversion for islet autoantibodies (against GAD, insulin, or insulinoma-associated antigen-2 [IA-2]) until diagnosis of diabetes. Enteroviral RNA in serum or rectal swabs was detected using reverse transcriptase PCR with primers specific for the conserved 5' noncoding region, detecting essentially all enterovirus serotypes., Results: Of 140 children who seroconverted to repeated positivity for islet autoantibodies at a median age of 4.0 years, 50 progressed to type 1 diabetes during a median follow-up of 4.2 years. The risk of progression to clinical type 1 diabetes in the sample interval following detection of enteroviral RNA in serum (three diabetes cases diagnosed among 17 intervals) was significantly increased compared with that in intervals following a negative serum enteroviral RNA test (33 cases diagnosed among 1,064 intervals; hazard ratio 7.02 [95% CI 1.95-25.3] after adjusting for number of autoantibodies). Results remained significant after adjustment for ZnT8-autoantibodies and after restriction to various subgroups. Enteroviral RNA in rectal swabs was not predictive of progression to type 1 diabetes. No evidence for viral persistence was found., Conclusions: This novel observation suggests that progression from islet autoimmunity to type 1 diabetes may increase after an enterovirus infection characterized by the presence of viral RNA in blood.

Objective: Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated beta-cell failure are unclear., Research Design and Methods: Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2-5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR., Results: Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor-bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-gamma and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including beta-cells and alpha-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells., Conclusions: These results strongly suggest the presence of a circuit for the destruction of beta-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-gamma and CXCL10 in beta-cells. CXCL10 secreted from beta-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-gamma in the islets, not only damaging beta-cells but also accelerating CXCL10 generation in residual beta-cells and thus further activating cell-mediated autoimmunity until all beta-cells have been destroyed.

Previous studies have suggested that enterovirus infections during pregnancy may increase the risk of type 1 diabetes in the offspring. Our aim was to evaluate the role of first trimester enterovirus infections in a larger cohort of pregnant women. Two series of pregnant women were analyzed as follows: 948 women (series 1) and 680 women (series 2) whose child developed clinical diabetes before the ages of 15 or 7 years, respectively. An equal number of control women with a nondiabetic child was selected. Acute enterovirus infections were diagnosed by measuring IgM class antibodies against coxsackievirus B5 (series 1) and a mixture of coxsackievirus B3, coxsackievirus A16, and echovirus 11 antigens (series 2). In series 2, all sera were also analyzed for IgG class antibodies against an enterovirus peptide antigen. In addition, 152 randomly selected case-control pairs and all IgM-positive mothers' sera were tested for enterovirus RNA (series 2). In series 1, 3.1% of case women had IgM antibodies against coxsackievirus B5 antigen compared with 4.1% of control women (NS). In series 2, 7.1% of case and 5.3% of control women had IgM against the mixture of enterovirus antigens (NS). IgG class enterovirus antibodies did not differ between the groups. Enterovirus RNA was found only in one case woman (0.3%) of the subgroup of samples and in 5.7% of 70 IgM-positive women. The results suggest that enterovirus infection during the first trimester of pregnancy is not associated with increased risk for type 1 diabetes in the child.

Previous studies suggest that enterovirus infections may initiate and accelerate beta-cell damage years before the clinical manifestation of type 1 diabetes. We have now analyzed the role of enterovirus infections in the initiation of autoimmunity in children who have tested positive for diabetes-associated autoantibodies in a prospective study starting at birth (the Finnish Diabetes Prediction and Prevention Study). The frequency of enterovirus infections was studied using both serology and testing for the presence of enterovirus RNA in the sera of 21 children who developed and retained autoantibodies and in 104 control subjects chosen from the same study cohort and matched for the time of birth, sex, and HLA alleles determining genetic diabetes susceptibility. Sample intervals were taken as basic units of follow-up, to which the observed number of infections was adjusted. Enterovirus infections were detected in 26% of sample intervals in the case subjects and in 18% of the sample intervals in the control children (P = 0.03). A temporal relationship between enterovirus infections and the induction of autoimmunity was found; enterovirus infections were detected in 57% of the case subjects during a 6-month follow-up period preceding the first appearance of autoantibodies compared with 31% of the matched control children in the same age-group (odds ratio 3.7, 95% CI 1.2-11.4). The frequency of adenovirus infections did not differ between the patient and control groups. Our data imply that enterovirus infections are associated with the development of beta-cell autoimmunity and provide evidence for the role of enteroviruses in the initiation of beta-cell destruction.

Enteroviruses have been examined for their possible role in the etiology of IDDM for nearly 40 years, yet the evidence remains inconclusive. The mechanism of acute cytolytic infection of beta-cells, proposed by earlier studies, appears to be incompatible with the long preclinical period of autoimmunity preceding IDDM. Advances in molecular biology have improved our understanding of enteroviral biology and of potential alternative pathogenic mechanisms through which enteroviruses may cause diabetes. The focus of future human studies will likely shift from people with IDDM to those with prediabetic autoimmunity to determine whether acute enteroviral infections can promote progression from autoimmunity to overt diabetes. We propose that such studies use assays to detect enteroviral RNA, in addition to IgM serology. RNA assays can overcome sensitivity and type-specificity limitations of IgM assays as well as identify diabetogenic strains of enteroviruses, if such exist. Evaluation of the role of enteroviruses in triggering beta-cell autoimmunity in humans will require large prospective studies of young children. The Diabetes Autoimmunity Study in the Young--one of very few such studies currently underway--is focusing on potential interactions between HLA class II genes and enteroviral infections. Future studies will likely examine interactions between viral infections and non-HLA IDDM candidate genes, including those that may determine beta-cell tropism of candidate viruses.

Coxsackievirus B infections have been associated with clinical manifestation of insulin-dependent diabetes mellitus (IDDM) in several studies, but their initiating role in the slowly progressing beta-cell damage is not known. This is the first prospective study designed to assess the role of coxsackie B and other enterovirus infections in the induction and acceleration of this process. Three separate series were studied: 1) an intrauterine exposure series comprising 96 pregnant mothers whose children subsequently manifested IDDM and 96 control mothers whose children remained nondiabetic; 2) a cohort of 22 initially unaffected siblings of diabetic children who were followed until they developed clinical IDDM (mean observation time, 29 months) and 110 control siblings who remained nondiabetic; 3) a case-control series comprising 90 children with newly diagnosed IDDM and 90 control subjects. Enterovirus infections were identified on the basis of significant increases in serum IgG, IgM, or IgA class antibodies against a panel of enterovirus antigens (capture radioimmunoassay). Enterovirus antibodies were significantly elevated in pregnant mothers whose children subsequently manifested IDDM, particularly in cases in which IDDM appeared at a very young age, before the age of 3 years (P < 0.005). Serologically verified enterovirus infections were almost two times more frequent in siblings who developed clinical IDDM than in siblings who remained nondiabetic (mean, 1.0 vs. 0.6 infections/follow-up year; P < 0.001). This difference was seen both close to the diagnosis of IDDM and several years before diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Infection of autoimmune New Zealand mice with the D variant of encephalomyocarditis (EMC) virus results in beta-cell damage and clinical diabetes. The induction of diabetes in parental NZB and NZW strains was independent of sex. However, the susceptibility to virus-induced diabetes in their F1 offspring was sex dependent. This susceptibility was significantly higher in male (NZB X NZW) F1 mice as compared with female F1 mice. Castration of male F1 mice significantly reduced the susceptibility to diabetes. These results suggest that parental NZB and NZW strains have recessive genes at different loci which do not allow sex hormones to influence the susceptibility to diabetes. It is concluded that both the genetic background of the host and sex hormones influence the development of virus-induced diabetes in autoimmune New Zealand mice.

The M variant of encephalomyocarditis virus (EMC) infects pancreatic beta cells and causes the development of a diabetic syndrome in susceptible strains of mice. By examining four F1 crosses of susceptible and resistant strains of mice, we found that the development of diabetes after infection was inherited as a recessive trait. Analysis of the data from the F2 generation indicated that more than one gene was involved in the development of EMC-diabetes. The severity and frequency of abnormal glucose levels in EMC-infected animals was markedly influenced by environmental factors.

DBA/2 and Balb/cBY mice were infected with approximately 30 plaque-forming units of the M-variant of encephalomyocarditis (EMC-M) virus. Seven days after inoculation the majority of the animals of both strains were hyperglycemic. A significant correlation between increased concentrations of virus in the pancreas and hyperglycemia was found among individual DBA/2 animals, but not among Balb/cBY mice. T-lymphocyte depletion of DBA/2 mice before infection failed to alter the incidence or severity of hyperglycemia in comparison to intact animals. Conversely, hyperglycemia in T-lymphocyte-depleted Balb/cBY mice was reduced substantially in comparison to infected immunocompetent animals. There appears to be at least two genetically influenced pathogenic mechanisms of diabetes in EMC-M virus-infected mice. In some strains of animals, hyperglycemia results exclusively from viral infection and the consequent injury to the beta cells, whereas in other animals, viral damage to the islets is compounded by immunologic events.

Adult, male ICR Swiss mice are susceptible to the diabetogenic effects of the D-variant of encephalomyocarditis virus (EMC-D) in contrast to adult C3H/HeJ male mice, which are relatively resistant. To date, experimental evidence suggests that the immune system plays a role in the pathogenesis of this infection. We have investigated the potential involvement of the immune system in the pathogenesis of EMC-D-induced diabetes using cyclosporin-A (CyA), a potent immunosuppressive drug. The data show that treatment with CyA results in increased severity and incidence of diabetes in susceptible ICR Swiss mice and induction of diabetes in resistant C3H/HeJ mice. It is concluded that immune mediation probably is not involved in the early pathogenesis of EMC-D-induced diabetes in mice.

Insulin-dependent diabetes mellitus (IDDM) results from the destruction of pancreatic beta cells. Viruses have been suggested as one of the possible causes. The evidence for viruses comes largely from experiments in animals, but several studies in humans also point to viruses as a trigger of this disease in some cases. Encephalomyocarditis (EMC) virus, Mengovirus (2T), and Coxsackie B4 virus infect and destroy pancreatic beta cells when inoculated into mice. This results in hypoinsulinemia and hyperglycemia. The development of EMC virus-induced diabetes is dependent on the genetic background of the host and genetic makeup of the virus. Animals with diabetes for several months show some long-term complications, including glomerulosclerosis, ocular changes, and decreased bone formation and mineralization in addition to acute metabolic changes. EMC virus-induced diabetes can be prevented by a live-attenuated vaccine. The capacity of Coxsackie B4 virus to induce diabetes is also influenced by the genetic background of the host. However, Mengovirus-induced diabetes is not dependent on the genetic background of the host. In contrast to the EMC, Mengo, and Coxsackie B4 viruses, reovirus type 1 seems to be somehow associated with an autoimmune response producing a diabetes-like syndrome in suckling mice. This virus produces an autoimmune polyendocrinopathy that results in very mild and transient glucose intolerance. Several common human viruses including mumps, Coxsackie B3 and B4 viruses, and reovirus type 3 can infect human beta cells in culture and destroy them. A variant of Coxsackie B4 virus has been isolated from the pancreas of a child who died of acute-onset IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible contribution of the immune system to the pathogenesis of virus-induced diabetes mellitus was investigated using the D-variant of encephalomyocarditis (EMC-D) virus. Studies on the F1 and backcross progeny of susceptible and resistant strains of mice gave no suggestion of a linkage between susceptibility and the major histocompatibility locus. Immuno-suppression by antilymphocyte serum did not prevent the induction of EMC-D-induced diabetes. Athymic nude mice infected with EMC-D virus showed a nearly identical diabetogenic response as compared with heterozygous littermates. Passive transfer of lymphocytes from mice made diabetic with EMC-D virus into normal mice failed to produce diabetes. From these and other studies, we conclude that the development of EMC-D-induced diabetes is due to the direct destruction of beta-cells by the virus and that the contribution of the immune response to the pathogenesis of this disease is, at the most, minor.

The induction of insulin-dependent diabetes in outbred male and female mice was examined using a combination of the usually nondiabetogenic B-variant of encephalomyocarditis (EMC-B) virus and single low doses of streptozocin (STZ). Neither EMC-B virus nor low doses of STZ were overtly diabetogenic when administered alone; however, when these two insults occurred 1 day apart, diabetes resulted in male but not in female mice. The induction of diabetes was dependent on the time interval between these two insults, since EMC-B virus and STZ given 4 days apart did not induce diabetes. Unexpectedly, when the order of these two insults was reversed, diabetes occurred. The absence of diabetes when EMC-B virus was given before STZ suggested the possibility that virus-induced interferon blocked the cytotoxic effects of STZ. This suggestion was supported by the observation that an antiserum against beta interferon abrogated the virus-mediated protection against STZ-mediated cytotoxicity. Also, Poly I:C administered before a single diabetogenic dose of STZ delayed the onset of severe hyperglycemia.