Enteroviral infection in human type 1 diabetes: correlative evidence from multiple tissue sources in nPOD samples

Background and aims: Considerable evidence has accumulated to support the hypothesis that type 1 diabetes (T1D) is associated with an enteroviral infection. Much of this depends on the analysis of viral infection in blood samples but definitive proof that this is also associated with the presence of enterovirus within the organs of affected individuals is still lacking. The purpose of this study was to undertake the first fully coordinated analysis of multiple tissues available within the JDRF\u2019s network of pancreatic organ donors with diabetes (nPOD) collection. These tissues were analysed using different methods, in multiple collaborating laboratories, to assess the correlation of potential indices of enteroviral infection. Materials and methods: Tissue samples (pancreas/ spleen/ PBMCs) from controls, T1D and autoantibody positive (AAb+) cases were prepared by the nPOD Pathology Core and distributed in a blinded manner to each laboratory. Samples were immunostained for enteroviral capsid protein VP1 and class I MHC [using IHC in FFPE sections and IF in frozen sections] and probed by in situ hybridisation for enteroviral genome. Enterovirus sequence analysis was attempted in extracts prepared from frozen spleen and PBMCs in a selection of the cases. Results: Expression of enteroviral capsid protein, VP1, and hyper-expression of class I MHC were detected consistently in the islets of Langerhans of patients with T1D while VP1 was also detected in the spleens. Immunopositivity was remarkably concordant between the different laboratories, despite the different methodologies employed. Tissues from a total of 101 cases were then examined and scored for several indices of viral infection including VP1 immunopositivity, class I MHC hyperexpression, positive in situ hybridisation signals and the presence of viral sequences. Among 28 controls, a total of 54 indices of viral infection were assessed independently and 7 were reported as positive (13%). In 52 type 1 diabetes cases (including those with residual insulin-containing islets and those without) 99 indices were scored and 51 were positive (51.5%). Among 21 autoantibody positive cases, 43 indices were assessed and 16 were positive (37.2%). PCR amplification and nucleic acid sequencing analysis of RNA extracted from cultured spleen cells or PBMCs revealed the presence of several enterovirus serotypes and the positivity correlated with VP1 staining in sections of tissue from the same samples. Conclusion: The results support the conclusion that enteroviral infection occurs at much higher frequency in T1D pancreas than in controls. Virus can be found both in blood samples and in other tissues (e.g. pancreas, spleen). Similar evidence of persisting virus was also found in AAb+ cases, supporting the hypothesis that enteroviruses may play a role early in the development of T1D