Characterisation of enterovirus RNA detected in the pancreas and other specimens of live patients with newly diagnosed type 1 diabetes in the DiViD study

Abstract

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study is the first study to laparoscopically collect pancreatic tissue and purified pancreatic islets together with duodenal mucosa, serum, peripheral blood mononuclear cells (PBMCs) and stools from six live adult patients (age 24-35 years) with newly diagnosed type 1 diabetes. The presence of enterovirus (EV) in the pancreatic islets of these patients has previously been reported.

Methods: In the present study we used reverse transcription quantitative real-time PCR (RT-qPCR) and sequencing to characterise EV genomes present in different tissues to understand the nature of infection in these individuals.

Results: All six patients were found to be EV-positive by RT-qPCR in at least one of the tested sample types. Four patients were EV-positive in purified islet culture medium, three in PBMCs, one in duodenal biopsy and two in stool, while serum was EV-negative in all individuals. Sequencing the 5' untranslated region of these EVs suggested that all but one belonged to enterovirus B species. One patient was EV-positive in all these sample types except for serum. Sequence analysis revealed that the virus strain present in the isolated islets of this patient was different from the strain found in other sample types. None of the islet-resident viruses could be isolated using EV-permissive cell lines.

Conclusions/interpretation: EV RNA can be frequently detected in various tissues of patients with type 1 diabetes. At least in some patients, the EV strain in the pancreatic islets may represent a slowly replicating persisting virus.

Keywords: Enterovirus; Laparoscopy; Organs; Pancreas; Persistent infection; RT-qPCR; Sequence; Type 1 diabetes; Viral RNA.

Fig. 1

Phylogenetic analysis of EV-positive samples based on the partial nucleotide sequence of the 5′UTR region (439–512 nucleotides in E30 GenBank accession no. KP266571). EVs can be divided into phylogenetic groups I and II using nucleotide sequence of this genome region. Virus obtained from the stool sample of patient 3 clusters in phylogroup I and all other viruses to phylogroup II. The same EV nucleotide sequence was present in all EV-positive samples from patient 6 except for the islet culture medium, which contained a different viral nucleotide sequence. Patients are identified by numbers 1–6, followed by the type of sample sequenced. Bootstrap values over 75 are marked in the phylogenetic tree. The distance scale is shown in the lower left corner. Islets, medium of cultured islets; Isolate, isolated virus; Pancreas R, RNAlater sample from pancreas; Pancreas S, snap-frozen sample from pancreas

Fig. 2

Sequence alignments of enterovirus strains detected in samples from patients with type 1 diabetes representing nucleotides 439–512 of the 5′UTR of E30 viral genome (GenBank accession no. AM237033). Patients are identified by numbers 1–6. Nucleotides that show variation to the consensus sequence (shown at the bottom of the figure) between virus strains are shown. Variable nucleotides between virus strains obtained from the same individual are marked with red rectangles. Islets, medium of cultured islets; Isolate, isolated virus; Pancreas R, RNAlater sample from pancreas; Pancreas S, snap-frozen sample from pancreas