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

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.

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/HYPOTHESIS: Enterovirus 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. METHODS: A 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. RESULTS: Vaccinated 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/INTERPRETATION: We 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 the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.

AIMS/HYPOTHESIS: Enteroviral 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. METHODS: Enteroviral 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. RESULTS: Clone 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/INTERPRETATION: When 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.

Detection of enterovirus RNA in pancreas and lymphoid tissues of organ donors with type 1 diabetes.

Aims/hypothesis: The nPOD-Virus group collaboratively applied innovative technologies to detect and sequence viral RNA in pancreas and other tissues from organ donors with type 1 diabetes. These analyses involved the largest number of pancreas samples collected to date. Methods: We analysed pancreas, spleen, pancreatic lymph nodes, and duodenum samples from the following donor groups: a) donors with type 1 diabetes (n=71), with (n=35) or without (n=36) insulin-containing islets, (b) donors with single or double islet autoantibody positivity without diabetes (n=22) and c) autoantibody-negative donors without diabetes (control donors) (n=74). Five research laboratories participated in this collaborative effort using approaches for unbiased discovery of RNA viruses (two RNA-Seq platforms), targeted detection of Enterovirus A-D species using RT-PCR, and tests for virus growth in cell-culture. Results: Direct RNA-Seq did not detect virus signal in pancreas samples, whereas RT-PCR detected enterovirus RNA confirmed by sequencing in low amounts in pancreas samples in three of the five donor groups, namely donors with type 1 diabetes with insulin-containing islets, 16% (5/32) donors being positive, donors with single islet autoantibody positivity with 53% (8/15) donors being positive, and non-diabetic donors with 8% (4/49) being enterovirus RNA positive. Detection of enterovirus RNA was significantly more frequent in single islet autoantibody-positive donors compared to donors with type 1 diabetes with insulin-deficient islets (p-value <0.001) and control donors (p-value 0.004). In some donors, pancreatic lymph nodes were also positive. RT-PCR detected enterovirus RNA also in spleen of a small number of donors and virus enrichment in susceptible cell lines before RT-PCR resulted in much higher rate in spleen positivity, particularly in donors with type 1 diabetes. Interestingly, the enterovirus strains detected did not cause a typical lytic infection, possibly reflecting their persistence-prone nature. Conclusions/interpretation: This was the largest coordinated effort to examine the presence of enterovirus RNA in pancreas of organ donors with type 1 diabetes, using a multitude of assays. These findings are consistent with the notion that both the subjects with type 1 diabetes and those with islet autoantibodies may carry a low-grade enterovirus infection in the pancreas and lymphoid tissues.

Serum 25-hydroxyvitamin D and fatty acids in relation to the risk of microbial infections in children: The TRIGR Divia study.

BACKGROUND & AIMS: Nutrient status may affect the risk of microbial infections and play a role in modulating the immune response against such infections. The aim of this study was to determine whether serum 25-hydroxyvitamin D [25(OH)D] and serum fatty acids in infancy are associated with microbial infections by the age of 18 months. METHODS: Altogether 576 newborn infants from Trial to Reduce IDDM in the Genetically at Risk (TRIGR) born between 2002 and 2007 were included. The concentration of 25(OH)D vitamin and proportions of 26 fatty acids (presented as % of total fatty acids) were analyzed in cord blood serum and in sera taken at 6, 12, and 18 months of age. The cord blood samples and mean of 6-18-month values were used as exposures. Infections were detected by screening IgG antibodies against 10 microbes using enzyme immunoassay and antibodies against 6 coxsackievirus B serotypes by plaque neutralization assay in serum samples taken at 18 months of age. RESULTS: A higher proportion of n-3 polyunsaturated fatty acids (PUFAs) and especially long-chain n-3 PUFAs at birth and at the age of 6-18 months was associated with decreased risk of coxsackievirus B2 infection unadjusted and adjusted for region, case-control status, and maternal type 1 diabetes. Higher proportion of docosapentaenoic acid (DPA, 22:5 n-3) at birth was associated with a decreased risk of respiratory syncytial virus infection. 25(OH)D vitamin concentration was not consistently associated with the risk of infections. When only infected children were included docosahexaenoic acid (DHA, 22:6 n-3) and arachidonic acid (20:4 n-6) proportions were positively associated with IgG antibody levels against influenza A virus. 25(OH)D vitamin concentration showed an inverse association with rotavirus IgG levels among children with rotavirus seropositivity. CONCLUSIONS: In young children with increased susceptibility to type 1 diabetes, long-chain n-3 PUFAs may influence the risk of viral infections and immune response against the infections. However, this association may depend on the type of virus suggesting virus-specific effects.

Expression of toll-like receptors in the pancreas of recent-onset fulminant type 1 diabetes.

Fulminant type 1 diabetes, established in 2000, is defined as a novel subtype of diabetes mellitus that results from remarkably acute and almost complete destruction of pancreatic beta cells at the disease onset. In this study, we aimed to clarify the pathogenesis of fulminant type 1 diabetes with special reference to insulitis and viral infection. We examined pancreatic autopsy samples from three patients who had died soon after the onset of disease and analyzed these by immunohistochemistry and in situ-hybridization. The results were that both beta and alpha cell areas were significantly decreased in comparison with those of normal controls. Mean beta cell area of the patients just after the onset was only 0.00256 % while that of normal control was 1.745 %. Macrophages and T cells-but no natural killer cells-had infiltrated the islets and the exocrine pancreas. Although both of them had massively infiltrated, macrophages dominated islet infiltration and were detected in 92.6 % of the patients' islets. Toll-like receptor (TLR) 3, a sensor of viral components, was detected in 84.7+/- 7.0 % of T cells and 62.7+/- 32.3 % of macrophages (mean+/- SD) in all three patients. TLR7 and TLR9 were also detected in the pancreas of all three patients. Enterovirus RNA was detected in beta-cell positive islets in one of the three patients by in situ-hybridization. In conclusion, our results suggest that macrophage-dominated insulitis rather than T cell autoimmunity contributes to beta cell destruction in fulminant type 1 diabetes.

Differential Detection of Encapsidated versus Unencapsidated Enterovirus RNA in Samples Containing Pancreatic Enzymes-Relevance for Diabetes Studies.

Using immunohistochemistry, enterovirus capsid proteins were demonstrated in pancreatic islets of patients with type 1 diabetes. Virus proteins are mainly located in beta cells, supporting the hypothesis that enterovirus infections may contribute to the pathogenesis of type 1 diabetes. In samples of pancreatic tissue, enterovirus RNA was also detected, but in extremely small quantities and in a smaller proportion of cases compared to the enteroviral protein. Difficulties in detecting viral RNA could be due to the very small number of infected cells, the possible activity of PCR inhibitors, and the presence-during persistent infection-of the viral genome in unencapsidated forms. The aim of this study was twofold: (a) to examine if enzymes or other compounds in pancreatic tissue could affect the molecular detection of encapsidated vs. unencapsidated enterovirus forms, and (b) to compare the sensitivity of RT-PCR methods used in different laboratories. Dilutions of encapsidated and unencapsidated virus were spiked into human pancreas homogenate and analyzed by RT-PCR. Incubation of pancreatic homogenate on wet ice for 20 h did not influence the detection of encapsidated virus. In contrast, a 15-min incubation on wet ice dramatically reduced detection of unencapsidated forms of virus. PCR inhibitors could not be found in pancreatic extract. The results show that components in the pancreas homogenate may selectively affect the detection of unencapsidated forms of enterovirus. This may lead to difficulties in diagnosing persisting enterovirus infection in the pancreas of patients with type 1 diabetes.

Enterovirus Infections Are Associated With the Development of Celiac Disease in a Birth Cohort Study.

Enterovirus and adenovirus infections have been linked to the development of celiac disease. We evaluated this association in children who developed biopsy-proven celiac disease (N = 41) during prospective observation starting from birth, and in control children (N = 53) matched for the calendar time of birth, sex, and HLA-DQ genotype. Enterovirus and adenovirus infections were diagnosed by seroconversions in virus antibodies in longitudinally collected sera using EIA. Enterovirus infections were more frequent in case children before the appearance of celiac disease-associated tissue transglutaminase autoantibodies compared to the corresponding period in control children (OR 6.3, 95% CI 1.8-22.3; p = 0.005). No difference was observed in the frequency of adenovirus infections. The findings suggest that enterovirus infections may contribute to the process leading to celiac disease.

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

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.

Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA).

Control RNA for RT-PCR applications was amplified by nucleic acid sequence based amplification (NASBA) using the NucliSens Basic Kit. This method was used to construct positive control RNA for enterovirus, insulin, and G-protein RT-PCR, and for interferon-alpha real-time RT-PCR. The primers were designed to amplify identical RNA from RNA templates, which differs from the usual NASBA procedure, where opposite strand RNA is amplified from the target. This "inverse NASBA" method is easy to use and it does not require any expensive special equipment. The amplification reaction is done using a water bath and detection of amplified product by agarose gel electrophoresis. Generated RNA fragments were 195-714 bases long, of positive polarity and the amount of RNA was sufficient for thousands of RT-PCR reactions depending on the sensitivity of the RT-PCR.

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.

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.

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.

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 ß-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.

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.

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.

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.