Project 04

Virome in T1D and viral infiltration of pancreatic islets

• Principial Investigators:
  • TUD: Barbara Ludwig & Michele Solimena
  • UZH: Alexandra Trkola & Michael Huebr
• Students:
  • TUD: Arvind Reddy Kandi (1st year-PhD student)

Background: Type 1 Diabetes (T1D) results from the autoimmune destruction of β-cells. Several viral infections, such as Coxsackievirus B (CVB), rotavirus, mumps, and cytomegalovirus, have been associated with type 1 diabetes (T1D) by triggering an autoimmune response, whereas it is suspected that other still unidentified viral infections may also promote autoimmunity and thus be linked with T1D. We (Zürich – Trkola/Huber group) have developed, optimized, and validated our own sample preparation and metagenomic virus sequencing platform for use in diagnostics. Metagenomic sequencing is a novel and complex technology that allows comprehensive detection of potentially all viral (and other) pathogens present in a clinical sample by amplification and high-throughput sequencing of all nucleic acids present. Data is analysed by an in-house bioinformatic pipeline to detected pathogen reads and assign them to viral species. An 
important focus of our research was the investigation of the virome in immunocompromised and transplanted patients and in clinical cases with unknown aetiology of infection. We propose now to use these technologies to investigate the virome status after the onset of T1D. A virus that has been most often implicated in pancreatic islet destruction and possibly in T1D is the positive-sense single-stranded RNA Coxsackievirus 5 (CVB5). We (Dresden – Solimena group) have demonstrated that infection of rodent insulinoma INS-1 and MIN6 cells as well as mouse islets with CVB5 does not inhibit glucose-stimulated biosynthesis of insulin granules, but reduces their stores by inducing the acidification and premature activation of cathepsins in the Trans-Golgi Network, thus leading to the proteolytic degradation of granule proteins prior to their 
sorting at into secretory granules. Remarkably, we found that a protein encoded by SARS-CoV2, also a positive-sense single-stranded RNA virus implicated in beta cell destruction and type 1 diabetes, alters the luminal pH within the early secretory pathway. Hence, it is possible that a shared property of RNA viruses is to alter the pH homeostasis along the secretory pathway of infected cells. Thus, we propose here to test this hypothesis by evaluating alterations in the function, gene expression, ultrastructure and most 
importantly pH homeostasis of primary mouse and human islets upon CVB5 infection. We (Dresden – Ludwig group) have extensive experience on isolating human, pig and mouse islets and run the only human islet transplantation centre in Germany. A virus that has been implicated in pancreatic islet destruction and possibly in T1D is the CVB5. We (Dresden – Solimena group) have demonstrated that CVB5 infection of rodent insulinoma INS-1 and MIN6 cells as well as mouse islets does not inhibit glucose-stimulated biosynthesis of insulin granules, but reduce their stores, conceivably by targeting granule proteins for destruction prior their conversion along the secretory pathway. We propose now to evaluate the effects of CVB5 infection on the function of primary mouse and human islets in vitro by focusing on alterations on islet and β-cell ultrastructure and multiomics. We (Dresden – Ludwig group) have extensive experience on isolating human, pig and mouse islets and run the only human islet transplantation center in Germany. The current approach that will be established for CVB5 infection can be also used to test any infection that may derive from the analysis of the circulating virome by the Trkola/Huber group.

Aims: 1) To characterize the virome and the anti-viral antibody responses of patients with type 1 Diabetes with the use of viral metagenomics sequencing and VirScan, 2) To evaluate the effect of CVB5 infection on islet and β-cell ultrastructure, multi-omics and cell survival.

Added value through the collaboration between Dresden & Zurich: The Dresden group has in depth experience in the isolation and functional characterization of human and animal pancreatic islets respectively. The Zürich-Trkola/Huber group are closely collaborating and have established the viral metagenomic sequencing (mNGS) for diverse clinical specimens. The Dresden group will establish an experimental pipeline to evaluate the effects of CVB5 infection on pancreatic islet function and survival. This experimental pipeline would be further used to assess the effects of other viruses, that will be identified by the Zürich group through the virome analysis on pancreatic islets.

Synergies: We will collaborate with projects 8, 9 to evaluate the virome in plasma of patients with post-infection syndromes and assess whether the virome is related to immune and metabolic response to dietary interventions. Additionally, we will interact with projects 3, 6, 8 to potentially screen the impact of the infections established in these models on mouse islet structure and function in relation to the metabolic state.