Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Persistent low-level variants in a subset of viral genes are highly predictive of poor outcome in immunocompromised patients with cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus is the most common and serious opportunistic infection after solid organ and haematopoietic stem cell transplantation. In this study, we used whole-genome cytomegalovirus data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced cytomegalovirus samples from 16 immunocompromised paediatric patients with persistent viraemia. 8/16 patients died of complications due to cytomegalovirus infection. We also sequenced samples from 35 infected solid organ adult recipients of whom one died with cytomegalovirus infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. NGS sequencing also revealed non-fixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with non-fixed variants in these patients. These genes formed a viral signature which discriminated patients with cytomegalovirus infection who died from those that survived with high accuracy (AUC=0.96). Lymphocyte numbers for a subset of patients showed no recovery post-transplant in the patients who died. CONCLUSIONS: We hypothesise that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T cell function, potentially identifying early, those patients requiring non-pharmacological interventions.

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube.

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Mpp5a- and Rab11a-mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose that these roles for both Mpp5a and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction-remodelling role of Mpp5a is not specific to dividing cells.

Genomics of Ocular Chlamydia trachomatis After 5 Years of SAFE Interventions for Trachoma in Amhara, Ethiopia.

BACKGROUND: To eliminate trachoma as a public health problem, the World Health Organization recommends the SAFE (surgery, antibiotics, facial cleanliness, and environmental improvement) strategy. As part of the SAFE strategy in the Amhara Region, Ethiopia, the Trachoma Control Program distributed >124 million doses of antibiotics between 2007 and 2015. Despite this, trachoma remained hyperendemic in many districts and a considerable level of Chlamydia trachomatis (Ct) infection was evident. METHODS: We utilized residual material from Abbott m2000 Ct diagnostic tests to sequence 99 ocular Ct samples from Amhara and investigated the role of Ct genomic variation in continued transmission of Ct. RESULTS: Sequences were typical of ocular Ct at the whole-genome level and in tissue tropism-associated genes. There was no evidence of macrolide resistance in this population. Polymorphism around the ompA gene was associated with village-level trachomatous inflammation-follicular prevalence. Greater ompA diversity at the district level was associated with increased Ct infection prevalence. CONCLUSIONS: We found no evidence for Ct genomic variation contributing to continued transmission of Ct after treatment, adding to evidence that azithromycin does not drive acquisition of macrolide resistance in Ct. Increased Ct infection in areas with more ompA variants requires longitudinal investigation to understand what impact this may have on treatment success and host immunity.

Post-Vaccination Coronavirus Disease 2019: A Case-Control Study and Genomic Analysis of 119 Breakthrough Infections in Partially Vaccinated Individuals.

BACKGROUND: Post-vaccination infections challenge the control of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We matched 119 cases of post-vaccination severe acute respiratory syndrome coronavirus 2 infection with BNT162b2 mRNA or ChAdOx1 nCOV-19 to 476 unvaccinated patients with COVID-19 (September 2020-March 2021) according to age and sex. Differences in 60-day all-cause mortality, hospital admission, and hospital length of stay were evaluated. Phylogenetic, single-nucleotide polymorphism (SNP), and minority variant allele (MVA) full-genome sequencing analysis was performed. RESULTS: Overall, 116 of 119 cases developed COVID-19 post-first vaccination dose (median, 14 days). Thirteen of 119 (10.9%) cases and 158 of 476 (33.2%) controls died (P < .001), corresponding to the 4.5 number needed to treat (NNT). Multivariably, vaccination was associated with a 69.3% (95% confidence interval [CI]: 45.8 to 82.6) relative risk (RR) reduction in mortality. Similar results were seen in subgroup analysis for patients with infection onset ≥14 days after first vaccination and across vaccine subgroups. Hospital admissions (odds ratio, 0.80; 95% CI: .51 to 1.28) and length of stay (-1.89 days; 95% CI: -4.57 to 0.78) were lower for cases, while cycle threshold values were higher (30.8 vs 28.8, P = .053). B.1.1.7 was the predominant lineage in cases (100 of 108, 92.6%) and controls (341 of 446, 76.5%). Genomic analysis identified 1 post-vaccination case that harbored the E484K vaccine-escape mutation (B.1.525 lineage). CONCLUSIONS: Previous vaccination reduces mortality when B.1.1.7 is the predominant lineage. No significant lineage-specific genomic changes during phylogenetic, SNP, and MVA analysis were detected.

Whole-genome Sequencing Provides Data for Stratifying Infection Prevention and Control Management of Nosocomial Influenza A.

BACKGROUND: Influenza A virus causes annual epidemics in humans and is associated with significant morbidity and mortality. Haemagglutinin (HA) and neuraminidase (NA) gene sequencing have traditionally been used to identify the virus genotype, although their utility in detecting outbreak clusters is still unclear. The objective of this study was to determine the utility, if any, of whole-genome sequencing over HA/NA sequencing for infection prevention and control (IPC) in hospitals. METHODS: We obtained all clinical samples from influenza (H1N1)-positive patients at the Great Ormond Street Hospital between January and March 2016. Samples were sequenced using targeted enrichment on an Illumina MiSeq sequencer. Maximum likelihood trees were computed for both whole genomes and concatenated HA/NA sequences. Epidemiological data was taken from routine IPC team activity during the period. RESULTS: Complete genomes were obtained for 65/80 samples from 38 patients. Conventional IPC analysis recognized 1 outbreak, involving 3 children, and identified another potential cluster in the haemato-oncology ward. Whole-genome and HA/NA phylogeny both accurately identified the previously known outbreak cluster. However, HA/NA sequencing additionally identified unrelated strains as part of this outbreak cluster. A whole-genome analysis identified a further cluster of 2 infections that had been previously missed and refuted suspicions of transmission in the haemato-oncology wards. CONCLUSIONS: Whole-genome sequencing is better at identifying outbreak clusters in a hospital setting than HA/NA sequencing. Whole-genome sequencing could provide a faster and more reliable method for outbreak monitoring and supplement routine IPC team work to allow the prevention of transmission.

Norovirus Transmission Dynamics in a Pediatric Hospital Using Full Genome Sequences.

Background: Norovirus is a leading cause of worldwide and nosocomial gastroenteritis. The study aim was to assess the utility of molecular epidemiology using full genome sequences compared to routine infection prevention and control (IPC) investigations. Methods: Norovirus genomes were generated from new episodes of norovirus at a pediatric tertiary referral hospital over a 19-month period (n = 182). Phylogeny identified clusters of related sequences that were verified using epidemiological and clinical data. Results: Twenty-four clusters of related norovirus sequences ("sequence clusters") were observed, including 8 previously identified by IPC investigations ("IPC outbreaks"). Seventeen sequence clusters (involving 77/182 patients) were corroborated by epidemiological data ("epidemiologically supported clusters"), suggesting transmission between patients. Linked infections were identified among 44 patients who were missed by IPC investigations. Thirty-three percent of norovirus sequences were linked, suggesting nosocomial transmission; 24% of patients had nosocomial infections from an unknown source; and 43% were norovirus positive on admission. Conclusions: We show there are frequent introductions of multiple norovirus strains with extensive onward nosocomial transmission of norovirus in a pediatric hospital with a high proportion of immunosuppressed patients nursed in isolation. Phylogenetic analysis using full genome sequences is more sensitive than classic IPC investigations for identifying linked cases and should be considered when investigating norovirus nosocomial transmission. Sampling of staff, visitors, and the environment may be required for complete understanding of infection sources and transmission routes in patients with nosocomial infections not linked to other patients and among patients with phylogenetically linked cases but no evidence of direct contact.

Randomized controlled trial of molnupiravir SARS-CoV-2 viral and antibody response in at-risk adult outpatients.

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not been elucidated in at-risk populations. Non-hospitalised participants within 5 days of SARS-CoV-2 symptoms randomised to receive molnupiravir (n = 253) or Usual Care (n = 324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated. Trial registration: ISRCTN30448031.

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

BACKGROUND: The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. RESULTS: A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. CONCLUSIONS: The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit.

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously.

A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5.

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 in vivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are "unlocked" by Ubp2-mediated deubiquitination.

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

Whole-genome sequencing of ocular Chlamydia trachomatis isolates from Gadarif State, Sudan.

BACKGROUND: Trachoma, caused by ocular Chlamydia trachomatis, is the leading infectious cause of blindness worldwide. Sudan first reported trachoma in the 1930s and has since been consistently endemic. Ocular C. trachomatis previously isolated from trachoma patients in Sudan in 1963 was antigenically identical to an isolate from Saudi Arabia (A/SA1). No contemporary ocular C. trachomatis whole genome sequences have been reported from Sudan. METHODS: This study sequenced twenty ocular C. trachomatis isolates to improve understanding of pathogen diversity in North-East Africa and examine for genomic variation specific to Sudan, possibly related to the persistence of trachoma in surveyed communities. High quality, whole genome sequences were obtained from 12/20 isolates. RESULTS: All isolates were serovar A and had tarP and trpA sequences typical of classical, ocular C. trachomatis isolates. The Sudanese isolates formed a closely related subclade within the T2-trachoma clade of C. trachomatis phylogeny distinct from geographically disparate ocular isolates, with little intra-population diversity. We found 333 SNPs that were conserved in Sudanese ocular isolates but rare compared to other ocular C. trachomatis populations, which were focused in two genomic loci (CTA0172-CTA0173 and CTA0482). CONCLUSIONS: Limited intra-population diversity and geographical clustering of ocular C. trachomatis suggests minimal transmission between and slow diversification within trachoma-endemic communities. However, diversity may have been higher pre-treatment in these communities. Over-representation of Sudan-specific SNPs in three genes suggests they may have an impact on C. trachomatis growth and transmission in this population.

Apoptotic cells induce dendritic cell-mediated suppression via interferon-gamma-induced IDO.

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-gamma expression by DC in association with apoptotic environments. The specific generation of IFN-gamma by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-gamma and IDO blockade demonstrated a role for IFN-gamma and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-gamma-dependent. Blocking transforming growth factor-beta (TGF-beta) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-gamma-induced IDO and TGF-beta.

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells.

The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability.