Demonstration of persistent enterovirus in the pancreas of diabetic mice by in situ polymerase chain reaction

Abstract

Background: Although Enterovirus (EV) do not persist in the tissue, which is essential to maintain autoimmunity, they have been associated as the cause of chronic autoimmunity in some cases of insulin dependent diabetes mellitus (IDDM). Convincing reports, demonstrating persistent EV infections in the pancreases, are rare. Objectives: To determine the role of EV in IDDM, a mouse model was tested and in situ polymerase chain reaction (ISPCR) developed. The major problem of ISPCR are the high amounts of non-specific staining. In the current study we developed an ISPCR protocol which minimised non-specific staining and allowed the accurate localisation of the viral RNA in the tissue. Study design: Five mice were infected with coxsackievirus group B4, sacrificed 7 weeks later and the pancreases were harvested. The EV nucleic acid were localised and detected in the pancreases by ISPCR. Results: In the current study non-specific staining of ISPCR, due to DNA repair and diffuse artefacts, were minimised and the EV nucleic acids were localised in the β cells of the endocrine pancreases in all five diabetogenic mice. Conclusion: This study demonstrates an association of viral RNA with the development of diabetes in mice and the usefulness of ISPCR to determine the role of EV in IDDM.