Establishing Evidence for Enterovirus Infection in Chronic Disease

Viruses have long been considered among potential environmental triggers of type 1 diabetes mellitus. Epidemiologic and seroprevalence studies have associated enterovirus infection with development of prediabetic autoimmunity and with the onset of clinical diabetes. Enterovirus infection has also been temporally correlated with disease onset by virus isolation or by detection of viral genome by reverse transcription-polymerase chain reaction (RT-PCR). For the large-scale prospective studies that are required to firmly establish a causal relationship between enterovirus infection and development of prediabetic autoimmunity or progression from autoimmunity to clinical diabetes, sensitive RT-PCR methods must be used to detect virus prior to the onset of diabetic symptoms. We have developed an RT-seminested PCR protocol to detect enteroviruses in clinical specimens. This method is approximately 10,000-fold more sensitive than conventional, single-amplification PCR. Further, we have developed molecular methods to rapidly and reliably identify enterovirus serotype, bypassing the cumbersome and often problematic neutralization test. The molecular serotyping approach will be valuable in examining the relationships between particular virus serotypes or genotypes and specific diseases.