Detection of enterovirus RNA in pancreas and lymphoid tissues of organ donors with type 1 diabetes

Aims/hypothesis

The nPOD-Virus group collaboratively applied innovative technologies to detect and sequence viral RNA in pancreas and other tissues from organ donors with type 1 diabetes. These analyses involved the largest number of pancreas samples collected to date.

Methods

We analysed pancreas, spleen, pancreatic lymph nodes, and duodenum samples from the following donor groups: a) donors with type 1 diabetes (n=71), with (n=35) or without (n=36) insulin-containing islets, (b) donors with single or double islet autoantibody positivity without diabetes (n=22) and c) autoantibody-negative donors without diabetes (control donors) (n=74). Five research laboratories participated in this collaborative effort using approaches for unbiased discovery of RNA viruses (two RNA-Seq platforms), targeted detection of Enterovirus A-D species using RT-PCR, and tests for virus growth in cell-culture.

Results

Direct RNA-Seq did not detect virus signal in pancreas samples, whereas RT-PCR detected enterovirus RNA confirmed by sequencing in low amounts in pancreas samples in three of the five donor groups, namely donors with type 1 diabetes with insulin-containing islets, 16% (5/32) donors being positive, donors with single islet autoantibody positivity with 53% (8/15) donors being positive, and non-diabetic donors with 8% (4/49) being enterovirus RNA positive. Detection of enterovirus RNA was significantly more frequent in single islet autoantibody-positive donors compared to donors with type 1 diabetes with insulin-deficient islets (p-value <0.001) and control donors (p-value 0.004). In some donors, pancreatic lymph nodes were also positive. RT-PCR detected enterovirus RNA also in spleen of a small number of donors and virus enrichment in susceptible cell lines before RT-PCR resulted in much higher rate in spleen positivity, particularly in donors with type 1 diabetes. Interestingly, the enterovirus strains detected did not cause a typical lytic infection, possibly reflecting their persistence-prone nature.

Conclusions/interpretation

This was the largest coordinated effort to examine the presence of enterovirus RNA in pancreas of organ donors with type 1 diabetes, using a multitude of assays. These findings are consistent with the notion that both the subjects with type 1 diabetes and those with islet autoantibodies may carry a low-grade enterovirus infection in the pancreas and lymphoid tissues.

Research in context

What is already known about this subject?

• Enterovirus infections are among the prime candidates for environmental triggers of type 1 diabetes.

• Pancreas (and other tissue) samples of subjects with type 1 diabetes have not been extensively studied for the presence of enterovirus RNA.

What is the key question?

• Can enterovirus RNA be detected in the pancreas and lymphoid tissues of individuals with and without type 1 diabetes?

What are the new findings?

• Enterovirus RNA can be detected in low amounts in the pancreas and lymphoid tissues using selected enterovirus-specific methods.

• Detection of enterovirus RNA in the pancreas was most frequent in prediabetic subjects.

• Enterovirus RNA was found also in pancreatic lymph nodes and in spleen where it was more frequently detected in donors with type 1 diabetes compared to non-diabetic donors, with properties previously observed in persistent infections.

How might this impact on clinical practice in the foreseeable future?

• The findings support the enterovirus - type 1 diabetes association and may have an effect on the primary and secondary prevention strategies towards the disease.