Detection of Enteroviruses in Tissue Samples - Methods and applications in type 1 diabetes

Type 1 diabetes (T1D) is a disease in which insulin-producing beta cells in the islets of Langerhans in the pancreas are destroyed by an autoimmune process. The events leading to the development of the disease are complex and still partially unclear. A long preclinical phase defined by the presence of the diabetes-associated autoantibodies in blood precedes the onset of the disease. Genetic predisposition determines the susceptibility to develop T1D, but also environmental factors, of which enterovirus infections are one of the most evident candidates, play an important role. Numerous epidemiological studies have shown the connection between enteroviruses and T1D, and the detection of these viruses directly in the pancreas and other organs of diabetic patients has given new evidence of the role of enteroviruses in disease development. The aim of this study was to develop new immunohistochemical and in situ hybridization methods for enterovirus detection, and to use these methods for the analysis of tissue samples of diabetic and prediabetic subjects to further assess the role of these viruses in the process leading to T1D. A panel of immunohistochemical and in situ hybridization methods was set-up for the detection of enteroviruses in tissue samples. Immunostaining with nine different antibodies or antibody combinations as well as in situ hybridization were optimized for formalin-fixed paraffin-embedded tissues, and immunostaining with seven different antibodies or antibody combinations for frozen tissues. The optimization and validation of the methods, as well as the evaluation of the ability of the hybridization probe and each antibody to detect different enterovirus serotypes, was carried out in enterovirusinfected cultured cells. Most antibodies as well as in situ hybridization worked well and proved to be a suitable tool for enterovirus detection in tissue samples. The presence of enteroviruses and certain immunological markers was analyzed using immunohistochemical and in situ hybridization methods and RT-PCR from a prediabetic child who had accidentally died and who had been repeatedly positive for islet cell antibodies. The morphology of the pancreas was normal, and the only marker of a possible beta cell-damaging process was the strong production of beta2-microglobulin in the islets. The child was also positive for enterovirus in the pancreatic islets in immunohistochemical staining with two different enterovirus-specific antibodies; however, in situ hybridization assay was negative. All follow-up serum samples taken preceding his death turned out to be enterovirus-negative in RT-PCR. The presence of enteroviruses in the small intestine of T1D patients was analyzed in two separate studies. In the first study, small intestinal biopsies of twelve T1D patients and ten controls were analyzed by immunohistochemistry with enterovirus VP1 proteinspecific antibody and in situ hybridization. Nine of the patients (75 %) and one of the control subjects (10 %) were enterovirus positive in either in situ hybridization or immunohistochemistry (p=0,004). The presence of the virus was also confirmed by RTPCR in some of the cases. In the second study, the same result was confirmed in a larger cohort which included intestinal biopsies from 39 T1D patients and 41 controls, as well as from 40 celiac disease patients. 74 % of the T1D patients were enterovirus-positive in in situ hybridization compared to 29 % of the control subjects (p<0,001), and the same trend was also seen in RT-PCR. The presence of enterovirus in in situ hybridization was also associated with the detection of inflammation markers in the duodenal mucosa. In conclusion, methods for enterovirus detection in tissue samples were set-up and optimized. The panel included several antibodies or antibody combinations and in situ hybridization assay which can be used for the detection of enteroviruses in both formalin-fixed paraffin-embedded and frozen tissue samples. These methods were applied in the analysis of pancreatic tissue of a prediabetic child, as well as in the examination of small intestine biopsy samples of T1D patients. The results suggest that enteroviruses can be detected in the pancreas and duodenum of T1D patients. These findings further strengthen the hypothesis of a pathogenic role of enteroviruses in the development of type 1 diabetes.