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. 2025 Jun;68(6):1226-1241.
doi: 10.1007/s00125-025-06401-x. Epub 2025 Mar 17.

Joint analysis of the nPOD-Virus Group data: the association of enterovirus with type 1 diabetes is supported by multiple markers of infection in pancreas tissue

Sarah J Richardson  1 , Teresa Rodriguez-Calvo  2 , Jutta E Laiho  3 , John S Kaddis  4 , Julius O Nyalwidhe  5 , Irina Kusmartseva  6 , Sofia Morfopoulou  7 , Joseph F Petrosino  8 , Vincent Plagnol  9 , Kathrin Maedler  10 , Margaret A Morris  5   11 , Jerry L Nadler  12   13 , Mark A Atkinson  14 , Matthias von Herrath  15 , Richard E Lloyd  8 , Heikki Hyoty  3   16   17 , Noel G Morgan  1 , Alberto Pugliese  18 ; nPOD-Virus Group
Affiliations

Joint analysis of the nPOD-Virus Group data: the association of enterovirus with type 1 diabetes is supported by multiple markers of infection in pancreas tissue

Sarah J Richardson et al. Diabetologia. 2025 Jun.

Abstract

Aims/hypothesis: Previous pathology studies have associated enterovirus infections with type 1 diabetes by examining the enterovirus capsid protein 1 (VP1) in autopsy pancreases obtained near diabetes diagnosis. The Network for Pancreatic Organ Donors with Diabetes (nPOD) has since obtained pancreases from organ donors with type 1 diabetes (with broad age and disease duration) and donors with disease-associated autoantibodies (AAbs), the latter representing preclinical disease. Two accompanying manuscripts from the nPOD-Virus Group report primary data from a coordinated analysis of multiple enterovirus indices. We aimed to comprehensively assess the association of multiple enterovirus markers with type 1 diabetes.

Methods: The nPOD-Virus Group examined pancreases from 197 donors, recovered between 2007 and 2019, classified into five groups: donors with type 1 diabetes, with residual insulin-containing islets (T1D-ICI group, n=41) or with only insulin-deficient islets (T1D-IDI, n=42); donors without diabetes who are AAb-negative (ND, n=83); and rare donors without diabetes expressing a single AAb (AAb+, n=22) or multiple AAbs (AAb++, n=9). We assessed the overall association of multiple indicators of enterovirus infection, case-by-case and between donor groups, as well as assay agreement and reproducibility, using various statistical methods. We examined data from 645 assays performed across 197 nPOD donors.

Results: Detection of enterovirus indices by independent laboratories had high reproducibility, using both enterovirus-targeted and unbiased methods. T1D-ICI donors had significantly higher (p<0.001) proportions of positive assay outcomes (58.4%) vs T1D-IDI (10.3%), ND (17.8%) and AAb-positive donors (AAb+ 24.6%; AAb++ 35.0%). Head-to-head comparisons revealed increased proportions of donors positive in two independent assays among T1D-ICI vs ND donors (VP1/HLA class I [HLA-I], p<0.0001; VP1/enterovirus-specific RT-PCR (EV-PCR), p=0.076; EV-PCR/HLA-I, p=0.016; proteomics/HLA-I, p<0.0001; VP1/proteomics, p=0.06). Among 110 donors examined for three markers (VP1, EV-PCR and HLA-I), 83.3% of T1D-ICI donors were positive in two or more assays vs 0% of ND (p<0.001), 26.7% of AAb+ (p=0.006), 28.6% of AAb++ (p=0.023) and 0% of T1D-IDI (p<0.001) donors.

Conclusions/interpretation: The nPOD-Virus Group conducted, to date, the largest and most comprehensive analysis of multiple indices of pancreatic enterovirus infections in type 1 diabetes; these were more prevalent in T1D-ICI and AAb++ donors than in other groups. Their preferential detection of these indices in donors with residual beta cells and autoimmunity implicates enterovirus infections across disease progression stages and supports a contribution to beta cell loss, directly or indirectly, even after diagnosis. The relatively small number of infected cells and the low amount of viral RNA support the existence of non-acute, low level, possibly persistent enterovirus infections in the pancreas.

Keywords: Autoimmunity; Enterovirus; Pancreas; Pancreatic beta cell; Pancreatic islet; Type 1 diabetes.

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Conflict of interest statement

Acknowledgements: The authors wish to thank all families associated with organ donation for research purposes whose gift was invaluable for this research. The authors are grateful to JDRF Program Officers who envisioned the formation of and provided programmatic guidance to the nPOD-Virus Group (R. Insel, J. Greenstein, T. Staeva, M. Nierras, J. Dunne). The authors thank all the members of the nPOD-Virus group. A list is included as electronic supplementary material (ESM). Data availability: Data generated and analysed during this study are available through the corresponding author upon request. Funding: This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by grants from JDRF/The Leona M. & Harry B. Helmsley Charitable Trust (3-SRA-2023-1417-S-B) and the Helmsley Charitable Trust (2018PG-T1D053, G-2108-04793). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at https://npod.org/for-partners/npod-partners/ . The nPOD-Virus Group was supported by Breakthrough T1D (formerly JDRF) grants (3-SRA-25-2012-516 and 3-SRA-2017-492-A-N awarded to AP). This work was supported by a Breakthrough T1D Career Development Award (5-CDA-2014-221-A-N) to SJR, an MRC Project Grant MR/P010695/1 awarded to SJR and NGM, and a Steve Morgan Foundation Grand Challenge Senior Research Fellowship awarded to SJR (22/0006504). The studies from the University of Exeter were supported by the National Institute for Health and Care Research Exeter Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Part of the studies were supported by the European Union program Frame Programme 7 (Persistent Virus Infection in Diabetes Network [PEVNET], contract no. 261441). JSK is also supported by a grant from the NIDDK-sponsored Human Islet Research Network (HIRN, RRID:SCR_014393; https://hirnetwork.org ; UC24 DK104162). KM was supported by a German Research Foundation grant (DFG). Authors’ relationships and activities: HH is a board member and stock owner of Vactech Oy, a Finnish biotech company that has contributed to the development of a CVB vaccine. The authors declare that there are no other relationships or activities that might bias, or be perceived to bias, their work. Contribution statement: SJR, TRC, JEL, JSK, REL, JON, JLN, IK, MAA, MvH, NGM, HH and AP made substantial contributions to the conception and design of the work. Assays were performed by SJR, TRC, JEL, JON, SM, KM, JFP and REL and interpretation of the individual specific assay experiments was performed by SJR, TRC, JEL, JON, REL, KM, MAM, VP, JFP, NGM, HH and AP. Formal combined analysis and interpretations were completed by SJR, TRC, JEL, JSK, JON, REL, HH and AP. MAM helped in sample selection, coordination, and procurement for proteomics assays, as well as editing of the manuscript. SJR, TRC, JEL, JSK and AP wrote the first draft of the manuscript. All authors reviewed critically the manuscript for important intellectual content and edited the manuscript. All authors have read and approved the final version of the manuscript. SJR, TRC, JL, JSK and AP are the guarantors of this work and, as such, have full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Figures

Fig. 1
Fig. 1
Summary of the types of assays performed and the number of donors assessed per assay or assay combination. Created in BioRender. Richardson, S. (2024) https://BioRender.com/k62i463. IF, immunofluorescence
Fig. 2
Fig. 2
Assessment of total assay positivity across different donor groups. The combination of all 645 assays performed over 197 nPOD donors demonstrates that T1D-ICI donors had a significantly higher proportion of positive assay outcomes when compared with ND, AAb+, AAb++ and T1D-IDI donors. *p<0.05, **p<0.01, ***p<0.001 (two-sided Fisher’s exact test; significant after false discovery rate [FDR] corrections for multiple comparisons). The total number of assays performed/donor group are shown within square brackets
Fig. 3
Fig. 3
Pairwise comparisons of different assays. Combinations of VP1 and EV-PCR, VP1 and HLA-I, EV-PCR and HLA-I, VP1 and proteomics, EV-PCR and proteomics and HLA-I and proteomics across different donor groups revealed that the T1D-ICI group had an increased percentage of donors who were double-positive (red) for the assays compared with ND donors. The number within each donut represents the total number of donors assessed in that donor group
Fig. 4
Fig. 4
Assessment of positivity in restricted EV-VP1, EV-PCR and HLA-I assays. (a) Evidence of autoimmunity and beta cell destruction is associated with an increase in the proportion of donors with positivity in two or more assays. Examination of the 110 donors in which three assays (EV-VP1, EV-PCR and HLA-I) were performed revealed that T1D-ICI donors are significantly more likely to have two or more positive assays when compared with ND, AAb+ and T1D-IDI donors. Donors with evidence of autoimmunity also had increased evidence of positivity in two or more assays when compared with ND donors. In T1D-IDI donors, the proportion with two or more assays positive was comparable to that seen in ND donors. *p<0.05, **p<0.01, ***p<0.001 (two-sided Fisher’s exact test; significant after false discovery rate [FDR] corrections for multiple comparisons). The block graphs demonstrate viral indicator counts in ND (n=36), AAb+ (n=15), AAb++ (n=7), T1D-ICI (n=30) and T1D-IDI (n=22) groups, with each square representing one individual. (b) Comparison of donors with or without evidence of autoimmunity, defined by the presence of circulating AAbs and/or insulitis (AI) with or without beta cells (β). No AAbs/insulitis and beta cells (n=40); AAbs/insulitis and beta cells (n=48), AAbs/insulitis and no beta cells (n=11), and no AAbs/insulitis and no beta cells (n=11). ***p<0.001 (two-sided Fisher’s exact test; significant after FDR corrections for multiple comparisons). The block graphs demonstrate viral indicator counts in individuals with no AAbs/insulitis and with beta cells (n=40), individuals with AAbs/insulitis and beta cells (n=48), individuals with AAbs/insulitis and no beta cells (n=11), and individuals with no AAbs/insulitis and no beta cells (n=11), with each square representing one individual. (c) Proportion of donors with two or more positive assays in whom three enterovirus-specific assays (VP1, EV-PCR and proteomics) were performed; ND (n=20), AAb+/++ (n=13) and T1D-ICIs (n=22) donors. The block graphs demonstrate viral indicator counts in ND (n=20), AAb+ (n=6), AAb++ (n=7), T1D-ICI (n=22) and T1D-IDI (n=2) donors. (d, e) Examination of donors in which four assays (VP1, PCR, HLA-I and proteomics; n=57) (d) and in which five assays (VP1, EV-PCR, proteomics, HLA-I and RNA-seq; n=34) (e) were performed revealed that T1D-ICI donors were significantly more likely to have two or more assays positive when compared with ND donors or those with evidence of islet autoimmunity (AAb+/++). *p<0.05, **p<0.01, ***p<0.001 (two-sided Fisher’s exact test). AI, AAbs and/or insulitis; β, beta cells
Fig. 5
Fig. 5
Sequential assessment of assay agreement. HLA-I assay results were evaluated sequentially by four other methods. The number of donors that tested positive (red) or negative (blue) for the assays listed on the x-axes are shown. Excluding the donors in the grey boxes, for whom data for certain assays were not available, (a) illustrates negative agreement and (b) shows positive agreement. As an example, in (a), from left to right, the negative agreement for each test being negative is reflected by the extent of the overlap in the blue colour. In this sequential analysis, the agreement is visualised across the different bars. As described in the main text, the highest agreement, whether negative or positive, was found between HLA-I hyperexpression and enterovirus VP1. No samples tested positive by all five assays. All donor groups were included in this analysis. Arrow indicates order of assessment. Prot., proteomics
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