Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Estimates of congenital cytomegalovirus-attributable infant mortality in high-income countries: A review.

As many as 5%-10% of infants with symptomatic congenital cytomegalovirus (cCMV) disease, or 0.4%-0.8% of all liveborn infants with cCMV infection, die in early infancy in high-income countries. However, estimates are uncertain due to several potential biases that can result from data limitations and study designs. First, infants with cCMV infections who die prior to diagnosis, which usually occurs at 1-4 weeks after birth, may be excluded from both the count of deaths and the denominator of cCMV births, resulting in left truncation and immortal time biases. These 'biases' are features of the data and do not reflect bias on the part of researchers, but understanding the potential existence of threats to validity can help with interpretation of findings. Left truncation of infant deaths occurring prior to diagnosis of cCMV can result in understatement of the burden of infant deaths due to cCMV. Conversely, overestimation of infant deaths associated with symptomatic cCMV may occur in clinical case series owing to greater representation of relatively severely affected infants owing to ascertainment and referral biases. In this review, we summarise the characteristics of 26 studies that reported estimates of cCMV-associated infant deaths, including potential biases or limitations to which those estimates may have been subject. We discuss study designs whose implementation might generate improved estimates of infant deaths attributable to cCMV. More complete estimates of the overall public health impact of cCMV could inform current and future screening, prevention, and vaccine research.

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.

Machine Learning Color Feature Analysis of a High Throughput Nanoparticle Conjugate Sensing Assay.

Plasmonic nanoparticles are finding applications within the single molecule sensing field in a "dimer" format, where interaction of the target with hairpin DNA causes a decrease in the interparticle distance, leading to a localized surface plasmon resonance shift. While this shift may be detected using spectroscopy, achieving statistical relevance requires the measurement of thousands of nanoparticle dimers and the timescales required for spectroscopic analysis are incompatible with point-of-care devices. However, using dark-field imaging of the dimer structures, simultaneous digital analysis of the plasmonic resonance shift after target interaction of thousands of dimer structures may be achieved in minutes. The main challenge of this digital analysis on the single-molecule scale was the occurrence of false signals caused by non-specifically bound clusters of nanoparticles. This effect may be reduced by digitally separating dimers from other nanoconjugate types. Variation in image intensity was observed to have a discernible impact on the color analysis of the nanoconjugate constructs and thus the accuracy of the digital separation. Color spaces wherein intensity may be uncoupled from the color information (hue, saturation, and value (HSV) and luminance, a* vector, and b* vector (LAB) were contrasted to a color space which cannot uncouple intensity (RGB) to train a classifier algorithm. Each classifier algorithm was validated to determine which color space produced the most accurate digital separation of the nanoconjugate types. The LAB-based learning classifier demonstrated the highest accuracy for digitally separating nanoparticles. Using this classifier, nanoparticle conjugates were monitored for their plasmonic color shift after interaction with a synthetic RNA target, resulting in a platform with a highly accurate yes/no response with a true positive rate of 88% and a true negative rate of 100%. The sensor response of tested single stranded RNA (ssRNA) samples was well above control responses for target concentrations in the range of 10 aM-1 pM.

Evidence supporting the use of therapeutic drug monitoring of ganciclovir in transplantation.

PURPOSE OF REVIEW: This review describes current knowledge of ganciclovir (GCV) and valganciclovir (ValGCV) pharmacokinetic/pharmacodynamic characteristics, highlighting the likely contribution from host genetic factors to interpatient variability. The evidence and challenges surrounding optimization of drug dosing through therapeutic drug monitoring (TDM) are examined, with recommendations made. RECENT FINDINGS: Pharmacokinetic studies of current dosing guidelines have shown high interindividual and intraindividual variability of GCV concentrations. This is sometimes associated with a slow decline in cytomegalovirus (CMV) viral load in some transplant recipients. A high incidence of GCV-associated myelosuppression has limited the use of this drug in the transplant setting. Patient groups identified to benefit from GCV TDM include pediatric patients, cystic fibrosis with lung transplantation, obese with kidney transplantation, and patients with fluctuating renal function or on hemodialysis. The emergence of refractory resistant CMV, particularly in immune compromised patients, highlights the importance of appropriate dosing of these antivirals. Host genetic factors need to be considered where recently, two host genes were shown to account for interpatient variation during ganciclovir therapy. Therapeutic Drug Monitoring has been shown to improve target antiviral-level attainment. The use of TDM may guide concentration-based dose adjustment, potentially improving virological and clinical outcomes. However, evidence supporting the use of TDM in clinical practice remains limited and further study is needed in the transplant cohort. SUMMARY: Further studies examining novel biomarkers are needed to guide target concentrations in prophylaxis and treatment. The use of TDM in transplant recipients is likely to improve the clinical efficacy of current antivirals and optimize outcomes in transplant recipients.

Detection of respiratory viruses directly from clinical samples using next-generation sequencing: A literature review of recent advances and potential for routine clinical use.

Acute respiratory infection is the third most frequent cause of mortality worldwide, causing over 4.25 million deaths annually. Although most diagnosed acute respiratory infections are thought to be of viral origin, the aetiology often remains unclear. The advent of next-generation sequencing (NGS) has revolutionised the field of virus discovery and identification, particularly in the detection of unknown respiratory viruses. We systematically reviewed the application of NGS technologies for detecting respiratory viruses from clinical samples and outline potential barriers to the routine clinical introduction of NGS. The five databases searched for studies published in English from 01 January 2010 to 01 February 2021, which led to the inclusion of 52 studies. A total of 14 different models of NGS platforms were summarised from included studies. Among these models, second-generation sequencing platforms (e.g., Illumina sequencers) were used in the majority of studies (41/52, 79%). Moreover, NGS platforms have proven successful in detecting a variety of respiratory viruses, including influenza A/B viruses (9/52, 17%), SARS-CoV-2 (21/52, 40%), parainfluenza virus (3/52, 6%), respiratory syncytial virus (1/52, 2%), human metapneumovirus (2/52, 4%), or a viral panel including other respiratory viruses (16/52, 31%). The review of NGS technologies used in previous studies indicates the advantages of NGS technologies in novel virus detection, virus typing, mutation identification, and infection cluster assessment. Although there remain some technical and ethical challenges associated with NGS use in clinical laboratories, NGS is a promising future tool to improve understanding of respiratory viruses and provide a more accurate diagnosis with simultaneous virus characterisation.

Viruses associated with acute respiratory infection in a community-based cohort of healthy New Zealand children.

Acute respiratory infections (ARIs) are a major cause of morbidity among children. Respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. The rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. Children otherwise healthy aged 1 to 8 years were prospectively enrolled in the study during two consecutive winters, when ARIs peak in New Zealand. Parents completed a daily symptom diary for 8 weeks, during which time they collected a nasal swab from the child for each clinical ARI episode. A further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. All samples were tested for 15 respiratory viruses commonly causing ARI using molecular multiplex polymerase chain reaction assays. There were 575 ARIs identified from 301 children completing the study, at a rate of 1.04 per child-month. Swabs collected during an ARI were positive for a respiratory virus in 76.8% (307 of 400), compared with 37.3% (79 of 212) of swabs collected during asymptomatic periods. The most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. All of these were significantly more likely to be detected during ARIs than asymptomatic periods. Parent-administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. Interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses.

Review of studies of severe acute respiratory syndrome related coronavirus-2 pathogenesis in human organoid models.

Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) is the cause of Covid-19 which was classified as a global pandemic in March 2020. The increasing global health and economic burden of SARS-CoV-2 has necessitated urgent investigations into the pathogenesis of disease and development of therapeutic and vaccination regimens. Human trials of vaccine and antiviral candidates have been undertaken, but basic pathogenetic studies are still required to inform these trials. Gaps in understanding of cellular infection by, and immunity to, SARS-CoV-2 mean additional models are required to assist in improved design of these therapeutics. Human organoids are three-dimensional models that contain multiple cell types and mimic human organs in ex vivo culture conditions. The SARS-CoV-2 virus has been implicated in causing not only respiratory injury but also injury to other organs such as the brain, liver and kidneys. Consequently, a variety of different organoid models have been employed to investigate the pathogenic mechanisms of disease due to SARS-CoV-2. Data on these models have not been systematically assembled. In this review, we highlight key findings from studies that have utilised different human organoid types to investigate the expression of SARS-CoV-2 receptors, permissiveness, immune response, dysregulation of cellular functions, and potential antiviral therapeutics.

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.

Receptors in host pathogen interactions between human cytomegalovirus and the placenta during congenital infection.

Cellular receptors in human cytomegalovirus (HCMV) mother to child transmission play an important role in congenital infection. Placental trophoblast cells are a significant cell type in placental development, placental functional processes, and in HCMV transmission. Different cells within the placental floating and chorionic villi present alternate receptors for HCMV cell entry. Syncytiotrophoblasts present neonatal Fc receptors that bind and transport circulating maternal immunoglobulin G across the placental interface which can also be bound to HCMV virions, facilitating viral entry into the placenta and foetal circulation. Cytotrophoblast express HCMV receptors including integrin-α1ß1, integrin-αVß3, epidermal growth factor receptor and platelet-derived growth factor receptor alpha. The latter interacts with HCMV glycoprotein-H, glycoprotein-L and glycoprotein-O (gH/gL/gO) trimers (predominantly in placental fibroblasts) and the gH/gL/pUL128, UL130-UL131A pentameric complex in other placental cell types. The pentameric complex allows viral tropism of placental trophoblasts, endothelial cells, epithelial cells, leukocytes and monocytes. This review outlines HCMV ligands and target receptor proteins in congenital HCMV infection.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

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.

Global epidemiology of nonpolio enteroviruses causing severe neurological complications: A systematic review and meta-analysis.

Enteroviruses are RNA viruses found as commensals in the human gut and respiratory system, which may cause a wide spectrum of disease. Enteroviruses may cause severe neurologic complications including acute flaccid paralysis (AFP) and encephalitis and are the most commonly diagnosed agents of viral meningitis. Outbreaks of more severe disease are often associated with particular genotypes, such as enterovirus-A71 causing rhombencephalitis and AFP. There are more than 300 described genotypes of human enterovirus, with overlaps in clinical phenotypes between genotypes, and uncertainty about which genotypes are more prevalent in neurological manifestations. A systematic review of observational studies was conducted to evaluate the most prevalent enterovirus genotypes causing AFP, encephalitis, and meningitis. The genotyping methods and sampling sites were compiled as secondary outcomes. Sources included MEDLINE, Embase (publications until January 2019), and references selected from included studies. Meta-analyses using a random effects model were performed to calculate the pooled proportion of enterovirus genotypes in each disease. Ninety-six publications met the eligibility criteria, comprising 3779 AFP cases, 1140 encephalitis cases, and 32 810 meningitis cases. Enterovirus-A71 was most frequently associated with AFP (pooled proportion 0.12, 95% CI, 0.05-0.20) and encephalitis (0.77, 95% CI, 0.61-0.91). Echovirus 30 (0.35, 95% CI, 0.27-0.42) was the most predominant genotype in meningitis cases. Genotypes were most commonly determined using VP1 RT- reverse transcription-polymerase chain reaction, and most samples assessed were cerebrospinal fluid. With the emergence of enteroviruses as an increasing cause of neurological diseases, surveillance and testing need to increase to identify the aetiology of the most common and most severe disorders.

IFCC interim guidelines on rapid point-of-care antigen testing for SARS-CoV-2 detection in asymptomatic and symptomatic individuals.

With an almost unremittent progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections all around the world, there is a compelling need to introduce rapid, reliable, and high-throughput testing to allow appropriate clinical management and/or timely isolation of infected individuals. Although nucleic acid amplification testing (NAAT) remains the gold standard for detecting and theoretically quantifying SARS-CoV-2 mRNA in various specimen types, antigen assays may be considered a suitable alternative, under specific circumstances. Rapid antigen tests are meant to detect viral antigen proteins in biological specimens (e.g. nasal, nasopharyngeal, saliva), to indicate current SARS-CoV-2 infection. The available assay methodology includes rapid chromatographic immunoassays, used at the point-of-care, which carries some advantages and drawbacks compared to more conventional, instrumentation-based, laboratory immunoassays. Therefore, this document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 aims to summarize available data on the performance of currently available SARS-CoV-2 antigen rapid detection tests (Ag-RDTs), providing interim guidance on clinical indications and target populations, assay selection, and evaluation, test interpretation and limitations, as well as on pre-analytical considerations. This document is hence mainly aimed to assist laboratory and regulated health professionals in selecting, validating, and implementing regulatory approved Ag-RDTs.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

BACKGROUND: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. METHODS: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. RESULTS: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. CONCLUSION: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Investigational Antiviral Therapy Models for the Prevention and Treatment of Congenital Cytomegalovirus Infection during Pregnancy.

Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation, lifelong disease, and, in severe cases, fetal or neonatal death. Placental infection with HCMV is the major mechanism of mother-to-child transmission (MTCT) and fetal injury. Thus, any pharmaceutical antiviral interference to reduce viral load may reduce placental damage, MTCT, and fetal disease. However, there is currently no licensed HCMV antiviral for use during pregnancy. In this study, aciclovir and the HCMV-specific antivirals letermovir, maribavir, and cidofovir were compared with ganciclovir for antiviral effects in model systems of pregnancy, including first-trimester TEV-1 trophoblast cell cultures and third-trimester ex vivo placental explant histocultures. HCMV-infected trophoblasts at 7 days postinfection (dpi) showed an EC50 of 21 µM for aciclovir, 0.0007 µM for letermovir, 0.11 µM for maribavir, and 0.29 µM for cidofovir, relative to 0.42 µM for ganciclovir. Antivirals added at 10 µM showed no cytotoxic effects and did not affect trophoblast cell proliferation (P > 0.9999). Multiple-round HCMV replication measured at 7 dpi showed letermovir, maribavir, and cidofovir treatment inhibited immediate early, early, and true late viral protein expression as assayed on Western blots. Antiviral treatment of HCMV-infected placental explants showed significant inhibition (P < 0.05) of viral replication with letermovir (83.3%), maribavir (83.6%), cidofovir (89.3%), and ganciclovir (82.4%), but not aciclovir (P > 0.9999). In ex vivo model systems, recently trialed HCMV antivirals letermovir and maribavir were effective at inhibiting HCMV replication. They partly fulfil requirements for use as safe and effective therapeutics during pregnancy to control congenital HCMV. Clinical trials of these newer agents would assist assessment of their utility in pregnancy.

Changes in pancreatic exocrine function in young at-risk children followed to islet autoimmunity and type 1 diabetes in the ENDIA study.

BACKGROUNDS: We aimed to monitor pancreatic exocrine function longitudinally in relation to the development of islet autoimmunity (IA) and type 1 diabetes (T1D) in at-risk children with a first-degree relative with T1D, who were followed prospectively in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. METHODS: Fecal elastase-1 (FE-1) concentration was measured longitudinally in 85 ENDIA children from median age 1.0 (IQR 0.7,1.3) year. Twenty-eight of 85 children (progressors) developed persistent islet autoantibodies at median age of 1.5 (IQR 1.1,2.5) years, of whom 11 went on to develop clinical diabetes. The other 57 islet autoantibody-negative children (non-progressors) followed similarly were age and gender-matched with the progressors. An adjusted linear mixed model compared FE-1 concentrations in progressors and non-progressors. RESULTS: Baseline FE-1 did not differ between progressors and non-progressors, or by HLA DR type or proband status. FE-1 decreased over time in progressors in comparison to non-progressors (Wald statistic 5.46, P = .02); in some progressors the fall in FE-1 preceded the onset of IA. CONCLUSIONS: Pancreatic exocrine function decreases in the majority of young at-risk children who progress to IA and T1D.

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.

Cytomegalovirus infection in day care centres: A systematic review and meta-analysis of prevalence of infection in children.

Maternofetal transmission of cytomegalovirus (CMV) is the most common infectious cause of congenital malformation in developed countries. Maternal infection often results from close contact with infected children, and this may occur in day care centres (DCCs). A systematic review of observational studies was conducted to examine the prevalence of CMV infection among children attending DCCs. Meta-analysis using the random effect model was performed for studies including controls. Sources included PubMed, EMBASE (until August 2018), and references from identified publications. Inclusion criteria were studies reporting CMV infection prevalence among childcare children aged less than 7 years of age. Controls were children without childcare exposure. CMV infection was defined as viral excretion detected by culture, polymerase chain reaction, or CMV seropositivity. Twenty-eight publications including 8347 participants met the eligibility criteria. The pooled prevalence of CMV infection among children in childcare from all studies was 32% (95% CI 23-41). Within case-controlled studies, prevalence among children attending DCCs was 34% (95% CI 25-44), whereas prevalence among those without childcare exposure was 22% (95% CI 15-30). Meta-analysis showed a significant association between DCC attendance and CMV infection (odds ratio 2.69, 95% CI 1.68-4.30; heterogeneity χ2 /df = 8; I2  = 84%, P < 0.00001). Attendance at DCCs is significantly associated with increased risk of childhood CMV infection. Prevention strategies to reduce risk of CMV infection of pregnant women and children should involve review of DCC exposure and consideration of preventative hygiene strategies.