ObjectiveTo determine whether infection with human enterovirus or adenovirus, both common intestinal viruses, predicts development of coeliac disease.DesignCase-control study nested within Norwegian birth cohort recruited between 2001 and 2007 and followed to September 2016.SettingNorwegian population.ParticipantsChildren carrying the HLA genotype DR4-DQ8/DR3-DQ2 conferring increased risk of coeliac disease.ExposuresEnterovirus and adenovirus detected using real time polymerase chain reaction in monthly stool samples from age 3 to 36 months.Main outcome measureCoeliac disease diagnosed according to standard criteria. Coeliac disease antibodies were tested in blood samples taken at age 3, 6, 9, and 12 months and then annually. Adjusted odds ratios from mixed effects logistic regression model were used to assess the relation between viral infections before development of coeliac disease antibodies and coeliac disease.ResultsAmong 220 children, and after a mean of 9.9 (SD 1.6) years, 25 children were diagnosed as having coeliac disease after screening and were matched to two controls each. Enterovirus was found in 370 (17%) of 2135 samples and was significantly more frequent in samples collected before development of coeliac disease antibodies in cases than in controls (adjusted odds ratio 1.49, 95% confidence interval 1.07 to 2.06; P=0.02). The association was restricted to infections after introduction of gluten. High quantity samples (>100 000 copies/μL) (adjusted odds ratio 2.11, 1.24 to 3.60; P=0.01) and long lasting infections (>2 months) (2.16, 1.16 to 4.04; P=0.02) gave higher risk estimates. Both the commonly detected enterovirus species Enterovirus A and Enterovirus B were significantly associated with coeliac disease. The association was not found for infections during or after development of coeliac disease antibodies. Adenovirus was not associated with coeliac disease.ConclusionsIn this longitudinal study, a higher frequency of enterovirus, but not adenovirus, during early childhood was associated with later coeliac disease. The finding adds new information on the role of viral infections in the aetiology of coeliac disease.

doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2 Key points Question: Are parechovirus infections associated with development of celiac disease in childhood? Findings: In this case-control study, nested in a cohort of children genetically at risk for celiac disease, a higher frequency of parechovirus gut infections (tested in monthly stool samples) were associated with later celiac disease. Coinfection with both parechovirus and enterovirus was associated with a markedly increased risk for later celiac disease. Meaning: The association observed between parechovirus and future celiac disease, suggests that these common enteric infections could play a role in celiac disease development. .

Objectives Direct genotyping of adenovirus or enterovirus from clinical material using polymerase chain reaction (PCR) followed by Sanger sequencing is often difficult due to the presence of multiple virus types in a sample, or due to varying efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids. Here we present a simple protocol for virus genotyping using massive parallel amplicon sequencing. Methods The protocol utilized a set of 16 tailed degenerate primers flanking the seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent addition of dual indices enabled simultaneous sequencing of 384 different samples on an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on remapping to a set of references representative of the presently known repertoire of virus types. Results After validation with known virus types, the sequencing method was applied on 301 adenovirus‐positive samples and 350 enterovirus‐positive samples from a longitudinally collected series of stools from 83 children aged 3 to 36 months. We detected 7 different adenovirus types and 27 different enterovirus types. There were 37 (6.2%) samples containing more than one genotype of the same viral genus. At least one dual infection was experienced by 23 of 83 (28%) of the children observed over the 3 years' observation period. Conclusions Amplicon sequencing with a multiplex set of degenerate primers seems to be a rapid and reliable technical solution for genotyping of large collections of samples where simultaneous infections with multiple strains can be expected.

Importance: Celiac disease is an increasingly common immune-mediated disorder. The potential role of infections in celiac disease development is not well characterized. Objective: To test whether two frequent enteric viruses, parechovirus and anellovirus, were associated with subsequent celiac disease. Our a priori hypothesis was that children who later developed celiac disease would have a higher frequency of parechovirus infections before transglutaminase 2 antibody development. Anellovirus testing was exploratory, as a potential marker of immune status. Design: Matched case-control design nested within a longitudinal birth cohort (the MIDIA study) of children at genetic risk for celiac disease. Setting: Children carrying the HLA genotype DR4-DQ8/DR3-DQ2, recruited at birth from the general population throughout Norway during 2001-2007. Participants: Of 220 genetically at-risk children tested for celiac disease, 25 fulfilled the case criteria. Each case was matched for follow-up time, birthdate, and county of residence with two randomly selected children free from celiac disease (controls) from the cohort. Exposures: Parechoviruses, the primary exposure, are infectious agents capable of replication at high virus loads. Anellovirus, previously proposed to reflect immune status, represent a ubiquitous viral exposure at low loads. Viruses were detected and quantified in monthly stool samples (collected from 3 through 35 months of age) using real-time PCR methods. Main outcome and measures: Celiac disease diagnosis according to ESPGHAN 2012 criteria. We retrospectively tested blood samples taken at age 3, 6, 9, and 12 months, and then annually to determine when transglutaminase 2 antibodies developed. Results: Parechovirus was detected in 222 of 2005 stool samples (11.1%), and was more frequent in samples from cases before developing transglutaminase 2 antibodies (adjusted odds ratio [aOR] 1.67, 95% CI 1.14-2.45, P=0.01). The odds ratio was higher when both parechovirus and enterovirus were positive in the same sample (aOR 4.73, 95% CI 1.26-17.67, P=0.02). Anellovirus was detected in 1540 of 1829 samples (84.2%). Anellovirus status did not differ significantly between case and control subjects. Conclusions and Relevance: Parechovirus infections in early life were associated with development of celiac disease in genetically at-risk children, suggesting a novel preventive target if confirmed in future studies.