Maarit Oikarinen

Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes

The Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3–9 weeks after onset of type 1 diabetes in six adult patients (age 24–35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh-frozen whole pancreatic tissue using PCR and sequencing. Nondiabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetic patients (two of nine controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (one of nine controls). Enterovirus-specific RNA sequences were detected in four of six patients (zero of six controls). The results were confirmed in various laboratories. Only 1.7% of the islets contained VP1+ cells, and the amount of enterovirus RNA was low. The results provide evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, which is consistent with the possibility that a low-grade enteroviral infection in the pancreatic islets contributes to disease progression in humans.

Enteroviruses in the pathogenesis of type 1 diabetes

The question if enteroviruses could cause beta-cell damage and type 1 diabetes has become more and more relevant when recent studies have provided new evidence supporting this scenario. One important observation is the recent discovery of IFIH1 as a risk gene for type 1 diabetes. This gene is an innate immune system receptor for enteroviruses offering one possible mechanism for the diabetogenic effect of enteroviruses. This is further emphasized by the observations suggesting that the innate immune system is activated in the pancreatic islets of type 1 diabetic patients and that the innate immune system is important for the defense against the virus and for the regulation of adaptive immune system. Important progress has also been gained in studies analyzing pancreas tissue for possible presence of enteroviruses. Several studies have found enteroviruses in the pancreatic islets of type 1 diabetic patients using various methods. The virus seems to be located in the islets while exocrine pancreas is mostly uninfected. One recent study found the virus in the intestinal mucosa in the majority of diabetic patients. Enteroviruses can also infect cultured human pancreatic islets causing either rapid cell destruction or a persistent-like noncytolytic infection. Combined with all previous, epidemiological findings indicating the risk effect of enteroviruses in cross-sectional and prospective studies, these observations fit to a scenario where certain diabetogenic enterovirus variants establish persistent infection in gut mucosa and in the pancreatic islets. This in turn could lead to a local inflammation and the breakdown of tolerance in genetically susceptible individuals. This is also supported by mouse experiments showing that enteroviruses can establish prolonged infection in the pancreas and intestine, and some virus strains cause beta-cell damage and diabetes. In conclusion, recent studies have strengthened the hypothesis that enteroviruses play a role in the pathogenesis of type 1 diabetes. These findings open also new opportunities to explore the underlying mechanism and get closer to causal relationship.

Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months’ observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.

Aims/hypothesisEnteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes.MethodsEnteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue.ResultsClone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1.Conclusions/interpretationWhen used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models

Aims/hypothesisEnterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus.MethodsA formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development.ResultsVaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice.Conclusions/interpretationWe conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.

Evaluation of immunohistochemistry and in situ hybridization methods for the detection of enteroviruses using infected cell culture samples

BACKGROUND Enterovirus infections are frequent in all age groups. In addition to acute infections, they have been connected to chronic diseases such as cardiomyopathies and type 1 diabetes. Based on this there is an increasing need for the reliable detection of enteroviruses in different kinds of tissue samples. OBJECTIVES The aim of this study was to set up a test panel which can detect a wide range of different enteroviruses in paraffin-embedded samples and fresh frozen samples using immunohistochemical and in situ hybridization methods. STUDY DESIGN A panel of nine enterovirus antibodies was optimized for the detection of different enterovirus types in both paraffin-embedded and frozen cell culture samples. In addition, an oligonucleotide probe detecting all human enteroviruses was evaluated for ISH in formalin-fixed paraffin-embedded cell culture samples. RESULTS Most antibodies worked well in both sample types. Some antibodies detected only one of the tested serotypes, whereas others detected several serotypes. ISH was able to detect all tested enterovirus types. CONCLUSIONS This test panel makes it possible to detect a wide range of different enterovirus types in both formalin-fixed paraffin-embedded and frozen samples. The same methods can also be applied for tissue sections, but may need further optimization for each tissue type.

Seroconversion to Islet Autoantibodies After Enterovirus Infection in Early Pregnancy

Gestational enterovirus (EV) infections have been associated with an increased risk for type 1 diabetes in the offspring. We therefore analyzed non-diabetic mothers for EV exposure in early pregnancy in relation to type 1 diabetes HLA-DQ risk genotypes and seroconversion to islet autoantibodies during pregnancy. Non-diabetic mothers who had islet autoantibodies (n=365) against glutamic acid decarboxylase (GADA), islet antigen-2 autoantibodies (IA-2A), or insulin autoantibodies (IAA), in early pregnancy and at delivery were compared to islet autoantibody-negative mothers (n=1457) matched for age and sampling date. Mothers were genotyped for HLA-DQ and analyzed for both EV-RNA and EV-IgM. EV-IgM, but not EV-RNA, was detected during early pregnancy in 12% of islet autoantibody-positive mothers compared to 11% of the controls. In early pregnancy, mothers with HLA-DQ 2/2 or 2/X genotypes showed an increased risk for islet autoantibodies at delivery (OR 1.85; p=0.001). After adjusting for parity, maternal age, year of birth, and season of early pregnancy, early pregnancy EV-IgM combined with DQ2/2 or 2/X increased the risk for islet autoantibodies (OR 3.10; 95% CI 1; p=0.008). EV-IgM in early pregnancy increased the risk for islet autoantibodies at delivery in non-diabetic mothers with HLA-DQ 2/2 or 2/X type 1 diabetes risk genotypes.

Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

BACKGROUND Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. OBJECTIVES This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. STUDY DESIGN A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10(-1) to 10(-8). Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. RESULTS RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10(-8)). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10(-7). The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). CONCLUSIONS All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

Detection of enterovirus in the islet cells of patients with type 1 diabetes: what do we learn from immunohistochemistry? Reply to Hansson SF, Korsgren S, Pontén F et al [letter]

Detection of enterovirus in the islet cells of patients with type 1 diabetes : what do we learn from immunohistochemistry? Reply to Hansson SF, Korsgren S, Ponten F et al [letter]

Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology.

BACKGROUND Enteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. OBJECTIVES The aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. STUDY DESIGN Four enterovirus RNA-targeted oligonucleotide RNA probes - two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) - were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. RESULTS The two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB -infected mice. CONCLUSIONS These novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.