Comparative genomics of Shiga toxin encoding bacteriophages

Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, lifethreatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as 24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of 24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, 24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however 24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The 24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potentia

Supported Decision-Making: Non-Domination Rather Than Mental Prosthesis

Recently, bioethicists and the UNCRPD have advocated for supported medical decision-making on behalf of patients with intellectual disabilities. But what does supported decision-making really entail? One compelling framework is Anita Silvers and Leslie Francis’ mental prosthesis account, which envisions supported decision-making as a process in which trustees act as mere appendages for the patient’s will; the trustee provides the cognitive tools the patient requires to realize her conception of her own good. We argue that supported decision-making would be better understood as a collaborative process, giving patients with intellectual disabilities the opportunity to make decisions in a respectful relationship with trusted others. We offer an alternative account of supported decision-making where the primary constraint is to protect the patient from domination by the trustee. This is advantageous in its preservation of the prospects for genuine collaboration, for the mental prosthesis approach ultimately reinforces a problematic ideal of isolated patient self-determination

454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases

Viromic Analysis of Wastewater Input to a River Catchment Reveals a Diverse Assemblage of RNA Viruses

Enteric viruses cause gastrointestinal illness and are commonly transmitted through the fecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes or emerging and invasive viral species. In this study, we investigated the use of viromics, i.e., high-throughput sequencing of the biosphere’s viral fraction, to detect human-/animal-pathogenic RNA viruses in the Conwy river catchment area in Wales, United Kingdom. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater and estuarine river water and sediment, followed by high-throughput RNA sequencing (RNA-Seq) analysis on the Illumina HiSeq platform, for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and we assembled a complete norovirus genotype GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional reverse transcription-quantitative PCR (qRT-PCR). The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested runoff from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included

Transcriptomic Analysis of Shiga-Toxigenic Bacteriophage Carriage Reveals a Profound Regulatory Effect on Acid Resistance in Escherichia coli

Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli

Gain-of-Sensitivity Mutations in a Trim5-Resistant Primary Isolate of Pathogenic SIV Identify Two Independent Conserved Determinants of Trim5α Specificity

Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5αQ, Trim5αTFP and Trim5CypA. Despite the high degree of amino acid identity between Trim5αQ and Trim5αTFP alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5αQ and Trim5αTFP alleles. While both alleles recognize the capsid surface, Trim5αQ and Trim5αTFP alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the β-hairpin. Given that none of the substitutions affected Trim5αQ alone, and the fact that the β-hairpin is conserved among retroviral capsids, we propose that the β-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5αTFP (without affecting Trim5αQ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid

Tracing the fate of wastewater viruses reveals catchment-scale virome diversity and connectivity

The discharge of wastewater-derived viruses in aquatic environments impacts catchment-scale virome composition and is a potential hazard to human health. Here, we used viromic analysis of RNA and DNA virus-like particle preparations to track virus communities entering and leaving wastewater treatment plants and the connecting river catchment system and estuary. We found substantial viral diversity and geographically distinct virus communities associated with different wastewater treatment plants. River and estuarine water bodies harboured more diverse viral communities in downstream locations, influenced by tidal movement and proximity to wastewater treatment plants. Shellfish and beach sand were enriched in viral communities when compared with the surrounding water, acting as entrapment matrices for virus particles. We reconstructed >40,000 partial viral genomes into 10,149 species-level groups, dominated by dsDNA and (+)ssRNA bacteriophages (Caudovirales and Leviviridae). We identified 73 (partial) genomes comprising six families that could pose a risk to human health; Astroviridae, Caliciviridae (sapovirus), Picornaviridae (cocksackievirus), Reoviridae (rotavirus), Parvoviridae and Circoviridae. Based on the pattern of viral incidence, we observe that wastewater-derived viral genetic material is commonly deposited in the environment, but due to fragemented nature of these viral genomes, the risk to human health is low, and is more likely driven by community transmission, with wastewater-derived viruses subject to cycles of dilution, enrichment and virion degradation influenced by local geography, weather events and tidal effects. Our data illustrate the utility of viromic analyses for wastewater- and environment-based epidemiology, and we present a conceptual model for the circulation of viruses in a freshwater catchment

Design as conversation with digital materials

This paper explores Donald Schön's concept of design as a conversation with materials, in the context of designing digital systems. It proposes material utterance as a central event in designing. A material utterance is a situated communication act that depends on the particularities of speaker, audience, material and genre. The paper argues that, if digital designing differs from other forms of designing, then accounts for such differences must be sought by understanding the material properties of digital systems and the genres of practice that surround their use. Perspectives from human-computer interaction (HCI) and the psychology of programming are used to examine how such an understanding might be constructed.</p