Respiratory infections and type 1 diabetes: Potential roles in pathogenesis.

Among the environmental factors associated with type 1 diabetes (T1D), viral infections of the gut and pancreas has been investigated most intensely, identifying enterovirus infections as the prime candidate trigger of islet autoimmunity (IA) and T1D development. However, the association between respiratory tract infections (RTI) and IA/T1D is comparatively less known. While there are significant amounts of epidemiological evidence supporting the role of respiratory infections in T1D, there remains a paucity of data characterising infectious agents at the molecular level. This gap in the literature precludes the identification of the specific infectious agents driving the association between RTI and T1D. Furthermore, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on the development of IA/T1D remains undeciphered. Here, we provide a comprehensive overview of the evidence to date, implicating RTIs (viral and non-viral) as potential risk factors for IA/T1D.

The virome in early life and childhood and development of islet autoimmunity and type 1 diabetes: A systematic review and meta-analysis of observational studies.

Viruses are postulated as primary candidate triggers of islet autoimmunity (IA) and type 1 diabetes (T1D), based on considerable epidemiological and experimental evidence. Recent studies have investigated the association between all viruses (the 'virome') and IA/T1D using metagenomic next-generation sequencing (mNGS). Current associations between the early life virome and the development of IA/T1D were analysed in a systematic review and meta-analysis of human observational studies from Medline and EMBASE (published 2000-June 2020), without language restriction. Inclusion criteria were as follows: cohort and case-control studies examining the virome using mNGS in clinical specimens of children ≤18 years who developed IA/T1D. The National Health and Medical Research Council level of evidence scale and Newcastle-Ottawa scale were used for study appraisal. Meta-analysis for exposure to specific viruses was performed using random-effects models, and the strength of association was measured using odds ratios (ORs) and 95% confidence intervals (CIs). Eligible studies (one case-control, nine nested case-control) included 1,425 participants (695 cases, 730 controls) and examined IA (n = 1,023) or T1D (n = 402). Meta-analysis identified small but significant associations between IA and number of stool samples positive for all enteroviruses (OR 1.14, 95% CI 1.00-1.29, p = 0.05; heterogeneity χ2  = 1.51, p = 0.68, I2  = 0%), consecutive positivity for enteroviruses (1.55, 1.09-2.20, p = 0.01; χ2  = 0.19, p = 0.91, I2  = 0%) and number of stool samples positive specifically for enterovirus B (1.20, 1.01-1.42, p = 0.04; χ2  = 0.03, p = 0.86, I2  = 0%). Virome analyses to date have demonstrated associations between enteroviruses and IA that may be clinically significant. However, larger prospective mNGS studies with more frequent sampling and follow-up from pregnancy are required to further elucidate associations between early virus exposure and IA/T1D.

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

Higher frequency of vertebrate-infecting viruses in the gut of infants born to mothers with type 1 diabetes.

BACKGROUND: Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing. METHODS: Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first-degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three-monthly in the first year of life. RESULTS: Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking. CONCLUSIONS: We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition.

Early-life factors contributing to type 1 diabetes.

The incidence of type 1 diabetes has increased since the mid-twentieth century at a rate that is too rapid to be attributed to genetic predisposition alone. While the disease can occur at any age, mounting evidence from longitudinal cohort studies of at-risk children indicate that type 1 diabetes associated autoantibodies can be present from the first year of life, and that those who develop type 1 diabetes at a young age have a more aggressive form of the disease. This corroborates the hypothesis that environmental exposures in early life contribute to type 1 diabetes risk, whether related to maternal influences on the fetus during pregnancy, neonatal factors or later effects during infancy and early childhood. Studies to date show a range of environmental triggers acting at different time points, suggesting a multifactorial model of genetic and environmental factors in the pathogenesis of type 1 diabetes, which integrally involves a dialogue between the immune system and pancreatic beta cells. For example, breastfeeding may have a weak protective effect on type 1 diabetes risk, while use of an extensively hydrolysed formula does not. Additionally, exposure to being overweight pre-conception, both in utero and postnatally, is associated with increased risk of type 1 diabetes. Epidemiological, clinical and pathological studies in humans support a role for viral infections, particularly enteroviruses, in type 1 diabetes, but definitive proof is lacking. The role of the early microbiome and its perturbations in islet autoimmunity and type 1 diabetes is the subject of investigation in ongoing cohort studies. Understanding the interactions between environmental exposures and the human genome and metagenome, particularly across ethnically diverse populations, will be critical for the development of future strategies for primary prevention of type 1 diabetes.

MicroRNAs in Type 1 Diabetes: Complex Interregulation of the Immune System, ß Cell Function and Viral Infections.

Since the discovery of the first mammalian microRNA (miRNA) more than two decades ago, a plethora of miRNAs has been identified in humans, now amounting to more than 2500. Essential for post-transcriptional regulation of gene networks integral for developmental pathways and immune response, it is not surprising that dysregulation of miRNAs is often associated with the aetiology of complex diseases including cancer, diabetes and autoimmune disorders. Despite massive expansion of small RNA studies and extensive investigation in diverse disease contexts, the role of miRNAs in type 1 diabetes has only recently been explored. Key studies using human islets have recently implicated virus-induced miRNA dysregulation as a pivotal mechanism of ß cell destruction, while the interplay between miRNAs, the immune system and ß cell survival has been illustrated in studies using animal and cellular models of disease. The role of specific miRNAs as major players in immune system homeostasis highlights their exciting potential as therapeutics and prognostic biomarkers of type 1 diabetes.

Enteroviruses and risk of islet autoimmunity or type 1 diabetes: systematic review and meta-analysis of controlled observational studies detecting viral nucleic acids and proteins.

BACKGROUND: Enteroviruses are routinely detected with molecular methods within large cohorts that are at risk of type 1 diabetes. We aimed to examine the association between enteroviruses and either islet autoimmunity or type 1 diabetes. METHODS: For this systematic review and meta-analysis, we searched PubMed and Embase for controlled observational studies from inception until Jan 1, 2023. Cohort or case-control studies were eligible if enterovirus RNA or protein were detected in individuals with outcomes of islet autoimmunity or type 1 diabetes. Studies in pregnancy or other types of diabetes were excluded. Data extraction and appraisal involved author contact and deduplication, which was done independently by three reviewers. Study quality was assessed with the Newcastle-Ottawa Scale and National Health and Medical Research Council levels of evidence. Pooled and subgroup meta-analyses were done in RevMan version 5.4, with random effects models and Mantel-Haenszel odds ratios (ORs; 95% CIs). The study is registered with PROSPERO, CRD42021278863. FINDINGS: The search returned 3266 publications, with 897 full texts screened. Following deduplication, 113 eligible records corresponded to 60 studies (40 type 1 diabetes; nine islet autoimmunity; 11 both), comprising 12077 participants (5981 cases; 6096 controls). Study design and quality varied, generating substantial statistical heterogeneity. Meta-analysis of 56 studies showed associations between enteroviruses and islet autoimmunity (OR 2·1, 95% CI 1·3-3·3; p=0·002; n=18; heterogeneity χ2/df 2·69; p=0·0004; I2=63%), type 1 diabetes (OR 8·0, 95% CI 4·9-13·0; p<0·0001; n=48; χ2/df 6·75; p<0·0001; I2=85%), or within 1 month of type 1 diabetes (OR 16·2, 95% CI 8·6-30·5; p<0·0001; n=28; χ2/df 3·25; p<0·0001; I2=69%). Detection of either multiple or consecutive enteroviruses was associated with islet autoimmunity (OR 2·0, 95% CI 1·0-4·0; p=0·050; n=8). Detection of Enterovirus B was associated with type 1 diabetes (OR 12·7, 95% CI 4·1-39·1; p<0·0001; n=15). INTERPRETATION: These findings highlight the association between enteroviruses and islet autoimmunity or type 1 diabetes. Our data strengthen the rationale for vaccine development targeting diabetogenic enterovirus types, particularly those within Enterovirus B. Prospective studies of early life are needed to elucidate the role of enterovirus timing, type, and infection duration on the initiation of islet autoimmunity and the progression to type 1 diabetes. FUNDING: Environmental Determinants of Islet Autoimmunity, European Association for the Study of Diabetes, JDRF, Australian National Health and Medical Research Council, and University of New South Wales.

Viruses and Type 1 Diabetes: From Enteroviruses to the Virome.

For over a century, viruses have left a long trail of evidence implicating them as frequent suspects in the development of type 1 diabetes. Through vigorous interrogation of viral infections in individuals with islet autoimmunity and type 1 diabetes using serological and molecular virus detection methods, as well as mechanistic studies of virus-infected human pancreatic ß-cells, the prime suspects have been narrowed down to predominantly human enteroviruses. Here, we provide a comprehensive overview of evidence supporting the hypothesised role of enteroviruses in the development of islet autoimmunity and type 1 diabetes. We also discuss concerns over the historical focus and investigation bias toward enteroviruses and summarise current unbiased efforts aimed at characterising the complete population of viruses (the "virome") contributing early in life to the development of islet autoimmunity and type 1 diabetes. Finally, we review the range of vaccine and antiviral drug candidates currently being evaluated in clinical trials for the prevention and potential treatment of type 1 diabetes.

SARS Coronavirus-2 Microneutralisation and Commercial Serological Assays Correlated Closely for Some but Not All Enzyme Immunoassays.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

Virus isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for diagnostic and research purposes.

Isolation of the new pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for diagnostic and research purposes including assessment of novel therapeutics. Several primary and continuous cell lines are currently used, and new organoid and engineered cell lines are being developed for improved investigation and understanding of the human immune response to this virus. Here we review the growth of SARS-CoV-2 in reference standard cell lines, engineered cell lines and new developments in this field.

Higher abundance of enterovirus A species in the gut of children with islet autoimmunity.

Enteroviruses (EVs) are prime candidate environmental triggers of islet autoimmunity (IA), with potential as vaccine targets for type 1 diabetes prevention. However, the use of targeted virus detection methods and the selective focus on EVs by most studies increases the risk for substantial investigation bias and an overestimated association between EV and type 1 diabetes. Here we performed comprehensive virome-capture sequencing to examine all known vertebrate-infecting viruses without bias in 182 specimens (faeces and plasma) collected before or at seroconversion from 45 case children with IA and 48 matched controls. From >2.6 billion reads, 28 genera of viruses were detected and 62% of children (58/93) were positive for ≥1 vertebrate-infecting virus. We identified 129 viruses as differentially abundant between the gut of cases and controls, including 5 EV-A types significantly more abundant in the cases. Our findings further support EV's hypothesised contribution to IA and corroborate the proposal that viral load may be an important parameter in disease pathogenesis. Furthermore, our data indicate a previously unrecognised association of IA with higher EV-A abundance in the gut of children and provide a catalog of viruses to be interrogated further to determine a causal link between virus infection and type 1 diabetes.

Distinct Gut Virome Profile of Pregnant Women With Type 1 Diabetes in the ENDIA Study.

BACKGROUND: The importance of gut bacteria in human physiology, immune regulation, and disease pathogenesis is well established. In contrast, the composition and dynamics of the gut virome are largely unknown; particularly lacking are studies in pregnancy. We used comprehensive virome capture sequencing to characterize the gut virome of pregnant women with and without type 1 diabetes (T1D), longitudinally followed in the Environmental Determinants of Islet Autoimmunity study. METHODS: In total, 61 pregnant women (35 with T1D and 26 without) from Australia were examined. Nucleic acid was extracted from serial fecal specimens obtained at prenatal visits, and viral genomes were sequenced by virome capture enrichment. The frequency, richness, and abundance of viruses were compared between women with and without T1D. RESULTS: Two viruses were more prevalent in pregnant women with T1D: picobirnaviruses (odds ratio [OR], 4.2; 95% confidence interval [CI], 1.0-17.1; P = .046) and tobamoviruses (OR, 3.2; 95% CI, 1.1-9.3; P = .037). The abundance of 77 viruses significantly differed between the 2 maternal groups (≥2-fold difference; P < .02), including 8 Enterovirus B types present at a higher abundance in women with T1D. CONCLUSIONS: These findings provide novel insight into the composition of the gut virome during pregnancy and demonstrate a distinct profile of viruses in women with T1D.

Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples.

More than 100 different enterovirus (EV) genotypes infect humans and contribute to substantial morbidity. However, current methods for characterisation of full-length genomes are based on Sanger sequencing of short genomic regions, which are labour-intensive and do not enable comprehensive characterisation of viral populations. Here, we describe a simple and sensitive protocol for the amplification and sequencing of near full-length genomes of human EV species using next generation sequencing. EV genomes were amplified from 89% of samples tested, with Ct values ranging between 15.7 and 39.3. These samples included 7 EV-A genotypes (CVA2, 5-7, 10, 16 and EV71), 19 EV-B genotypes (CVA9, CVB1-6, ECHO3, 4, 6, 7, 9, 11, 16, 18, 25, 29, 30, and EV69), 3 EV-C genotypes (CVA19 and PV2, 3) and 1 EV-D genotype (EV70). We characterised 70 EVs from 58 clinical stool samples and eight reference strains, with a minimum of 100X depth. We found evidence of co-infection in four clinical specimens, each containing two distinct EV genotypes (CVB3/ECHO7, CVB3/ECHO18 and ECHO9/30). Characterisation of the complete genome provided conclusive genotyping of EVs, which can be applied to investigate the intra-host virus evolution of EVs, and allows further identification and investigation of EV outbreaks.