Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes

Abstract

Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family.With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.

Enteroviruses and T1D: Is It the Virus, the Genes or Both which Cause T1D

“…Because of several such limitations to the detection of enteroviruses, we have previously established an adapted method to target single RNA molecules with short (~20 nucleotides) fluorescently labeled oligonucleotides in situ. Probes consist of a mixture of 40 short oligonucleotides covering the whole length of the viral genome and anneal to common regions of the RNA genome of the coxsackievirus family [ 77 ]. This enables targeting single RNA molecules.…”

“…Short labeled oligo RNA probes are more resistant to RNAse, and RNA detection is less affected by target RNA degradation and fragmentation. Through the availability of the well-characterized cohort of human pancreatic donor tissue established by nPOD [ 78 ], viral mRNA can be detected in the T1D pancreas with high sensitivity, specificity and accuracy and at lower viral loads than by classical immunostaining and even PCR [ 77 , 79 ]. Further ongoing studies of pancreas sections revealed remarkable significance of viral RNA expression in T1D pancreata, compared to controls without T1D [ 80 ].…”

Innate Viral Sensor MDA5 and Coxsackievirus Interplay in Type 1 Diabetes Development

“…In some reports, patients with T1D have detectable anti-enteroviral antibodies and CVB RNA in the blood and stool 6-12 months before autoantibody development [86,87]. Moreover, some studies have shown that recent onset patients with T1D have detectable enterovirus infections within their pancreata and islets [34,[88][89][90]. Patients with fulminant T1D have robust expression of MDA5, RIG-I, and the major capsid protein of enteroviruses, VP1, in both β-cells and α-cells within the islets compared to long-standing diabetic patients and non-diabetic controls [22].…”

“…The coxsackievirus type A (CVA) group has 24 serotypes; this group causes flaccid paralysis and severe infection [31,32]. The coxsackievirus type B (CVB) group has six serotypes, all correlated with T1D, known for causing spastic paralysis with mild infections [33][34][35][36][37][38][39][40][41][42][43][44][45]. CVB infection is transmitted by the fecal-oral route or by direct contact with mucosal secretions.…”

Enteroviral infections in the pathogenesis of type 1 diabetes: new insights for therapeutic intervention

“…Importantly, recent studies of unique pancreas biopsy samples from Norwegian patients with T1D (the DiViD samples [ 11 ]) have provided strong evidence for both the presence of HEV and enhanced islet anti-viral responses in newly-diagnosed patients [ 12 , 13 • , 14 • , 15 ]. In addition, ever more sensitive technologies are being developed to detect or interrogate viral infection and anti-viral responses in blood [ 8 , 16 •• , 17 , 18 , 19 , 20 ], islets [ 12 , 21 • , 22 • ], stool [ 9 ] and other tissues [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. These are currently being applied in new collaborative studies involving multiple laboratories who are employing differing expertise and complementary technologies to examine blinded tissue samples available from the network of Pancreatic Organ Donors with Diabetes (nPOD).…”