Genetic Determinants of Enterovirus Infections: Polymorphisms in Type 1 Diabetes and Innate Immune Genes in the MIDIA Study.

Enteroviruses have been suggested as triggers of type 1 diabetes (T1D). We aimed to assess whether established T1D susceptibility single nucleotide polymorphisms (SNPs) and candidate SNPs in innate immune genes were associated with the frequency of enterovirus infection in otherwise healthy children. Fifty-six established T1D SNPs and 97 other candidate immunity SNPs were typed in 419 children carrying the T1D high-risk genotype, HLA-DR4-DQ8/DR3-DQ2 genotype, and 373 children without this genotype. Enteroviral RNA was detected using real-time polymerase chain reaction, with primers detecting essentially all enterovirus serotypes, in 7,393 longitudinal stool samples collected monthly (age range 3-36 months). The most significant association was with two T1D SNPs, rs12150079 (ZPBP2/ORMDL3/GSDMB region) (enterovirus frequency: AA 7.3%, AG 8.7%, GG 9.7%, RR = 0.86, overall p = 1.87E-02) and rs229541 (C1QTNF6/SSTR3/RAC2) (enterovirus frequency: CC 7.8%, CT 9.7%, TT 9.4%, RR = 1.13, overall p = 3.6E-02), followed by TLR8 (rs2407992) (p = 3.8E-02), TLR3 (1914926) (p = 4.9E-02), and two other T1D SNPs (IFIH1 rs3747517, p = 4.9E-02 and PTPN22, rs2476601, p = 5.3E-02). However, the quantile-quantile plot of p-values with confidence intervals for all 153 SNPs did not reveal clear evidence for rejection of the complete null hypothesis. Among a number of SNPs in candidate genes, we found no evidence for strong associations with enterovirus presence in stool samples from Norwegian children.

Enterovirus RNA in longitudinal blood samples and risk of islet autoimmunity in children with a high genetic risk of type 1 diabetes: the MIDIA study.

AIMS/HYPOTHESIS: Only a few longitudinal molecular studies of enterovirus and islet autoimmunity have been reported, and positive results seem to be limited to Finland. We aimed to investigate an association between enterovirus RNA in blood and islet autoimmunity in the MIDIA study from Norway, a country which largely shares environmental and economic features with Finland. METHODS: We analysed serial blood samples collected at ages 3, 6, and 9 months and then annually from 45 children who developed confirmed positivity for at least two autoantibodies (against insulin, GAD65 and IA-2) and 92 matched controls, all from a cohort of children with a single high-risk HLA-DQ-DR genotype. Enterovirus was tested in RNA extracted from frozen blood cell pellets, using real-time RT-PCR with stringent performance control. RESULTS: Out of 807 blood samples, 72 (8.9%) were positive for enterovirus. There was no association between enterovirus RNA and islet autoimmunity in samples obtained strictly before (7.6% cases, 10.0% controls, OR 0.75 [95% CI 0.36, 1.57]), or strictly after the first detection of islet autoantibodies (10.5% case, 5.8% controls, OR 2.00 [95% CI 0.64, 6.27]). However, there was a tendency towards a higher frequency of enterovirus detection in the first islet autoantibody-positive sample (15.8%) compared with the corresponding time point in matched controls (3.2%, OR 8.7 [95% CI 0.97, 77]). Neither of these results was changed by adjusting for potential confounders, restricting to various time intervals or employing various definitions of enterovirus positivity. CONCLUSIONS/INTERPRETATION: Positivity for enterovirus RNA in blood did not predict the later induction of islet autoantibodies, but enterovirus tended to be detected more often at the islet autoantibody seroconversion stage.

Enterovirus RNA in peripheral blood may be associated with the variants of rs1990760, a common type 1 diabetes associated polymorphism in IFIH1.

OBJECTIVE: Polymorphisms in the IFIH1 (common rs1990760 and four rare rs35667974, rs35337543, rs35744605, rs35732034) have been convincingly associated with type 1 diabetes. The encoded protein (interferon-induced helicase C domain-containing protein 1) senses double-stranded RNA during replication of Picornavirales, including Enterovirus, a genus suspected in the etiology of type 1 diabetes. We therefore investigated whether the polymorphisms are associated with differences in the frequency of enterovirus RNA in blood. RESEARCH DESIGN AND METHODS: The study included 1001 blood samples, each from a child participating in the Norwegian 'Environmental Triggers of Type 1 Diabetes: the MIDIA study'. The enterovirus RNA was tested using qualitative semi-nested real-time reverse transcriptase PCR on RNA extracted from frozen cell packs after removal of plasma. Stool samples previously analyzed for enterovirus RNA were available in 417 children. RESULTS: The genotypes of IFIH1 rs1990760 were associated with different frequencies of enterovirus RNA in blood (7.0%, 14.4% and 9.5% bloods were enterovirus positive among children carrying the Ala/Ala, Ala/Thr and Thr/Thr genotypes, respectively, p = 0.012). This association remained essentially unchanged after adjustment for age and calendar year. The presence of enterovirus in the concomitantly sampled stool further increased the likelihood of enterovirus RNA in blood (odds ratio 2.40, CI 95% 1.13-4.70), but did not affect the association with IFIH1 rs1990760. The rare polymorphisms (individually, or pooled) were not significantly associated with enterovirus RNA in blood. CONCLUSIONS: The common IFIH1 SNP may modify the frequency of enterovirus RNA in blood of healthy children. This effect can help explain the association of IFIH1 with type 1 diabetes.

HLA-DRB1-DQA1-DQB1 genotype and frequency of enterovirus in longitudinal monthly fecal samples from healthy infants.

Enterovirus infections may be involved in the etiology of type 1 diabetes (T1D), which is strongly associated with certain human leukocyte antigen (HLA) class II haplotypes. Our aim was to assess whether HLA genotypes conferring varying degrees of risk for T1D were associated with enterovirus gut infections. From the general Norwegian population, 190 healthy infants at high-risk for T1D (DR4-DQ8/DR3-DQ2), and 383 infants without this genotype were identified. Non-DR4-DQ8/DR3-DQ2 genotypes were further categorized as conferring either an increased-to-moderate risk (DR4-DQ8 or DR3-DQ2), were protective (DQB1*06:02), or were neutral (all other genotypes). A total of 4626 monthly fecal samples taken between age 3 and 12 mo were tested for enterovirus RNA using real-time PCR. Enterovirus prevalence was 11.5% among high-risk children, and 12.2% in other children (adjusted odds ratio: 1.23, p=0.12). The prevalence was 11.3% in those with increased-to-moderate risk, 13.0% in the protective group, and 12.6% in the neutral group (likelihood ratio test, 3 d.f.: p=0.37). In conclusion, there was no statistically significant association between HLA genotype and the occurrence of human enterovirus gut infections.

Polymorphisms in the innate immune IFIH1 gene, frequency of enterovirus in monthly fecal samples during infancy, and islet autoimmunity.

Interferon induced with helicase C domain 1 (IFIH1) senses and initiates antiviral activity against enteroviruses. Genetic variants of IFIH1, one common and four rare SNPs have been associated with lower risk for type 1 diabetes. Our aim was to test whether these type 1 diabetes-associated IFIH1 polymorphisms are associated with the occurrence of enterovirus infection in the gut of healthy children, or influence the lack of association between gut enterovirus infection and islet autoimmunity.After testing of 46,939 Norwegian newborns, 421 children carrying the high risk genotype for type 1 diabetes (HLA-DR4-DQ8/DR3-DQ2) as well as 375 children without this genotype were included for monthly fecal collections from 3 to 35 months of age, and genotyped for the IFIH1 polymorphisms. A total of 7,793 fecal samples were tested for presence of enterovirus RNA using real time reverse transcriptase PCR.We found no association with frequency of enterovirus in the gut for the common IFIH1 polymorphism rs1990760, or either of the rare variants of rs35744605, rs35667974, rs35337543, while the enterovirus prevalence marginally differed in samples from the 8 carriers of a rare allele of rs35732034 (26.1%, 18/69 samples) as compared to wild-type homozygotes (12.4%, 955/7724 samples); odds ratio 2.5, p = 0.06. The association was stronger when infections were restricted to those with high viral loads (odds ratio 3.3, 95% CI 1.3-8.4, p = 0.01). The lack of association between enterovirus frequency and islet autoimmunity reported in our previous study was not materially influenced by the IFIH1 SNPs.We conclude that the type 1 diabetes-associated IFIH1 polymorphisms have no, or only minor influence on the occurrence, quantity or duration of enterovirus infection in the gut. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection.

Self-reported lower respiratory tract infections and development of islet autoimmunity in children with the type 1 diabetes high-risk HLA genotype: the MIDIA study.

AIM: To test whether self-reported lower respiratory tract infections in early infancy predicted risk for islet autoimmunity in genetically predisposed children. METHODS: The environmental triggers for type 1 diabetes (MIDIA) study recruited newborns in Norway to identify those with the human leukocyte antigen high-risk genotype DR4-DQ8/DR3-DQ2. Of 46 939 newborns genotyped, 1003 (2.1%) carried the high-risk genotype, of whom 885 children were followed longitudinally with questionnaires and blood samples for autoantibody testing at 3, 6, 9 and 12 months of age, and then annually until 4 years of age. The endpoint (autoimmunity) was defined as positivity for at least one of three autoantibodies (to insulin, glutamic acid decarboxylase (GAD) or protein tyrosine phosphatase-like protein (IA2)) on at least two consecutive samples. The parents responded in the questionnaires, whether the child had had 'pneumonia, bronchitis or respiratory syncytial virus'. Cox proportional hazards regression models with time-dependent covariates were used to estimate hazard ratios for autoimmunity using STATA 10. RESULTS: Forty-two children developed autoimmunity, of whom 15 later developed type 1 diabetes. For 17 of the 42 cases (40%) 'pneumonia, bronchitis or respiratory syncytial virus' was reported (0.5-4 years of age) before or at the onset of autoimmunity. For 187 of the 843 non-cases (22%) 'pneumonia, bronchitis or respiratory syncytial virus' was reported in the same age group. The hazard ratio was 3.4 (p=0.001, 95% confidence interval: 1.6-7.1) for developing autoimmunity. The estimated hazard ratio was only marginally influenced by adjustment for potential confounding factors. No association was found for other infectious self-reported symptoms. CONCLUSION: Self-reported lower respiratory tract infections were associated with increased risk of islet autoimmunity in early infancy.

Human enterovirus RNA in monthly fecal samples and islet autoimmunity in Norwegian children with high genetic risk for type 1 diabetes: the MIDIA study.

OBJECTIVE: To test whether the frequency of human enterovirus RNA in fecal samples collected monthly from early infancy was associated with development of multiple islet autoantibodies in children with the highest risk HLA genotype. RESEARCH DESIGN AND METHODS: Individuals carrying the HLA DRB1*0401-DQA1*03-DQB1*0302/DRB1*03-DQA1*05-DQB1*02 genotype were identified at birth and followed with monthly stool samples from age 3 to 35 months. Blood samples taken at age 3, 6, 9, and 12 months and then annually were tested for autoantibodies to insulin, GAD 65 and IA-2. Among 911 children, 27 developed positivity for two or more islet autoantibodies in two or more consecutive samples (case subjects). Two control subjects per case subject were matched by follow-up time, date of birth, and county of residence. Stool samples were analyzed for enterovirus with a semiquantitative real-time RT-PCR. RESULTS: The frequency of human enterovirus RNA in stool samples from case subjects before seroconversion (43 of 339, 12.7%) did not differ from the frequency in control subjects (94 of 692, 13.6%) (P = 0.97). Results remained essentially unchanged after adjustment for potential confounders, restriction to various time windows before seroconversion, or infections in the 1st year of life or after inclusion of samples collected after seroconversion. There was no difference in the average quantity of enterovirus RNA or in the frequency of repeatedly positive samples. The estimated relative risk for islet autoimmunity per enterovirus RNA-positive sample during follow-up (nested case-control analysis) was 1.12 (95% CI 0.66-1.91). CONCLUSIONS: There was no support for the hypothesis that fecal shedding of enteroviral RNA is a major predictor of advanced islet autoimmunity.

Predictors of sub-clinical enterovirus infections in infants: a prospective cohort study.

BACKGROUND: Enterovirus infections are common, although most often sub-clinical. The present purpose was to assess the impact of breastfeeding and other factors on enterovirus infections in infancy. METHODS: A prospective observational study was carried out on a population-based cohort of 639 Norwegian infants aged 3-12 months. The outcome was enterovirus RNA measured in monthly stool samples. Data on underlying determinants, such as dietary feeding and household factors, were reported in parental questionnaires. Multivariable logistic regression was performed to allow for common confounders. Statistical analyses were performed by GLLAMM using Stata 9.2, which corrects for subject-specific random effects. RESULTS: The prevalence of enterovirus in stools was 11.1% (475/4279). Risk of enterovirus infection decreased with increasing number of daily breastfeeds; the effect was most pronounced at the age of 3 months [odds ratio (OR), 0.85; 95% confidence interval (CI) 0.8-0.9, P < 0.001], gradually declining thereafter, reaching no effect at 11 months. Increased risk was associated with having one or more sibling(s) (OR 1.89; 95% CI 1.2-3.0), particularly if they attended daycare (OR 2.46; 95% CI 1.4-4.2), and with increasing exposure to other children (OR 1.04; 95% CI 1.0-1.1). There was a tendency towards higher prevalence of infection when a household's drinking water came from a well, and a protective effect of owning a dog or cat. CONCLUSIONS: Several factors may modify the risk for enterovirus infections in the first year of life. This study supports the protective effect of breastfeeding. The protection decreased with age and increased with dose of ingested milk.

Longitudinal observation of parechovirus in stool samples from Norwegian infants.

Parechoviruses are assumed to be common infectious agents, but their epidemiologic and pathogenic properties are not well known. The aim of the present study was to assess the prevalence and molecular epidemiology of Parechovirus in Norwegian infants, as well as to investigate whether the presence of virus correlated with symptoms of infection. A group of 102 infants was longitudinally followed: 51 infants with a high genetic risk for type 1 diabetes (aged 3-35 months), and 51 children without this genotype (aged 3-12). Stool samples were obtained each month, and symptoms of infection were recorded regularly on questionnaires. Human parechovirus was detected in 11.3% of 1,941 samples examined by real-time RT-PCR. There was a distinct seasonality, peaking from September to December. By 12 months of age, 43% of the infants had had at least one infection, while 86% of the infants had encountered the virus by the end of the second year. Based on the VP1 sequence, human parechovirus 1 was the most prevalent type (76%), followed by human parechovirus 3 (13%), human parechovirus 6 (9%), an unclassified human parechovirus (1%), and human parechovirus 2 (1%). Ljungan virus, a murine parechovirus, was examined with a separate real-time RT-PCR, but no virus was detected. There was no significant association between infections and the following symptoms: coughing, sneezing, fever, diarrhea or vomiting. In conclusion, human parechovirus infects frequently infants at an early age without causing disease.

Islet autoantibody development during follow-up of high-risk children from the general Norwegian population from three months of age: design and early results from the MIDIA study.

We describe the design of the MIDIA study and present serial islet autoantibody data from 3 months of age in the 526 first enrolled children from the general population carrying the type 1 diabetes high-risk HLA-DRB1*0401-DQA1*03-DQB1*0302/DRB1*0301-DQA1*05-DQB1*02 genotype. Blood samples were obtained from children at ages 3, 6, 9 and 12 months and annually thereafter to a median age of 12 months. Autoantibodies to insulin, glutamic acid decarboxylase and insulinoma-associated antigen-2 were measured with radiobinding assays. About 25,000 general population newborns were genotyped, and among 526 children with the high-risk HLA genotype, 2104 samples were assayed. Fourteen children were positive in at least two consecutive samples, including 12 who were positive for > or =2 autoantibodies at least once, of which five developed type 1 diabetes at median age 15.3 months. Seven of 14 persistently positive children seroconverted before 9 months, including two before 6 months of age. The estimated cumulative probability of multiple autoantibody positivity at 5 years was 7.3% (95% confidence interval: 3.5-12.4%). Thus, persistent islet autoimmunity is not uncommon in the first year of life in children from the general population carrying the high-risk HLA genotype, and may develop as early as at 6 months of age.

Asymptomatic circulation of HEV71 in Norway.

Widespread circulation of human enterovirus 71 was discovered in a prospective study of fecal samples obtained from healthy Norwegian children. Molecular characterization of the virus determined that it belonged to genotype C1. Complete sequencing of this strain, HEV71 804/NO/03, revealed differences in the 5'UTR and polymerase with respect to more pathogenic genotypes that may explain its reduced neurovirulence.

High prevalence of human enterovirus a infections in natural circulation of human enteroviruses.

Human enterovirus (HEV) infections can be asymptomatic or cause only mild illness; recent evidence may implicate HEV infection in type 1 diabetes mellitus and myocarditis. Here, we report the molecular characterization of HEV obtained in serial monthly collections from healthy Norwegian infants. A total of 1,255 fecal samples were collected from 113 healthy infants beginning at age 3 months and continuing to 28 months. The samples were analyzed for HEV nucleic acid by real-time PCR. Fifty-eight children (51.3%) had HEV infections. One hundred forty-five positive samples were typed directly by nucleotide sequencing of the VP1 region. HEV-A was detected most frequently, with an overall prevalence of 6.8%. HEV-B was present in 4.8% of the samples and HEV-C in only 0.2% of the samples. No poliovirus or HEV-D group viruses were detected. Twenty-two different serotypes were detected in the study period: the most common were EV71 (14.5%), CAV6 (10.5%), CAV4 (8.9%), E18 (8.9%), and CBV3 (7.3%). These findings suggest that the prevalence of HEV infections in general, and HEV-A infections in particular, has been underestimated in epidemiological studies based on virus culture.

DNA extraction and HLA genotyping using mailed mouth brushes from children.

The need for blood samples in genetic epidemiologic studies often leads to low response rate among non-diseased individuals, and the collection of blood samples is costly and labor-intensive. We tested the feasibility of extracting DNA for human leukocyte antigen (HLA) typing from buccal cells collected with mailed, self-administered mouth brushes. A random sample of 1474 Norwegian children aged 0-17 yr was contacted by mail and received information about the study and mouth brushes for buccal cell samples. Brushes were returned by mail, DNA was extracted and the HLA-DQA1 and -DQB1 allelic polymorphisms were determined using polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes. Mouth swabs were returned from 1068 (72.5%). Of these, DNA was extracted and HLA typing successfully completed for 1056 individuals (98.9%). In conclusion, we have described an efficient and safe set of methods for application in genetic epidemiologic studies of type 1 diabetes and other HLA-related diseases. A large proportion of randomly selected children returned self-administered mouth swabs with DNA of sufficient quality and quantity for HLA genotyping.