Biology subjects

Dewar, R.

Publications and source records attributed to Dewar, R..

5 recordsLinked to original sources

bioRxiv2025

Clinical performance evaluation of a tiling amplicon panel for whole genome sequencing of respiratory syncytial virus

Accurate genomic characterization of respiratory syncytial virus (RSV) is crucial for studies of epidemiology and viral evolution, and monitoring potential escape from newly authorized vaccines and antivirals. We adapted a viral whole genome tiling amplicon panel (UW-ARTIC) and developed a custom bioinformatic pipeline for high-throughput, cost-effective sequencing of RSV-A and RSV-B. We established genome acceptability criteria and determined the performance characteristics of the panel including assay sensitivity, specificity, breadth of genome recovery, accuracy, and precision using contrived and remnant clinical specimens. High-quality genomes (>95% genome completeness; >500X and >1000X average depth for whole genome and fusion gene respectively) were recovered from samples with Ct [≤] 30 ([~]594 and 2,004 copies per reaction for RSV-A and RSV-B respectively). Minor variants were accurately identified in sample mixtures of 5:95 and higher. The assay showed high accuracy when compared against Sanger, shotgun metagenomic, and hybridization capture-based sequencing; and high repeatability and reproducibility. The UW-ARTIC RSV panel has utility in genomic surveillance, clinical and research applications. It has been used to generate FDA-reportable data for clinical trials of RSV antiviral products, with robust performance characteristics in samples from around the globe from as recently as the 2023/24 season. Continued genomic surveillance and future updates to primer sets will be essential for continued recovery of genomes as RSV continues to evolve.

genomics↗

bioRxiv2024

ARTIC RSV amplicon sequencing reveals global RSV genotype dynamics.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections (LTRIs) in young children and adults over 65, contributing significantly to global healthcare burdens. With the recent approval of multiple pharmacological interventions for RSV, there is an increased demand for efficient, high-throughput sequencing methods to monitor RSV genetic diversity and any potential impact these interventions may have. Here we introduce two novel amplicon-based sequencing schemes designed for RSV A and B, optimised for integration with widespread existing ARTIC sequencing workflows. We demonstrate that these primer schemes can produce high quality genomes from RSV samples across the globe, with eight laboratories in five countries generating complete genomes on both Nanopore and Illumina sequencing platforms. The ability to effectively multiplex these RSV A and B primer schemes, enables streamlined, high-throughput sequencing without prior subtyping. Furthermore, these results provide a snapshot of the circulating diversity of RSV. Phylogenetic analysis of the 882 samples sequenced for this study suggests only minimal geographic clustering of RSV sequences, underscoring the global nature of RSV spread. It also highlights the distinct lineage dynamics seen between RSV A and B. This study represents an advancement in RSV genomics, providing robust tools for global sequencing efforts aimed at tracking RSV evolution and assessing the efficacy of new therapeutic interventions both rapidly and at scale.

genomics↗

bioRxiv2021

Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1

Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naive patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Forster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H. ImportanceA nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

microbiology↗

bioRxiv2019

Constitutive TRIM22 expression within the respiratory tract identifies tissue-specific and cell-type dependent intrinsic immune barriers to influenza A virus infection

We hypothesized that increased expression of antiviral host factors at portals of viral entry may protect exposed tissues from the constant threat of invading pathogens. Comparative transcriptomic analysis identified the broad-acting restriction factor TRIM22 (TRIpartite Motif 22) to be among the most abundantly expressed antiviral host factors in the lung, a major portal of entry for many respiratory pathogens. This was surprising, as TRIM22 is currently considered to be an interferon stimulated gene (ISG) product that confers protection following the activation of pathogen-induced cytokine-mediated innate immune defences. Using human respiratory cell lines and the airways of rhesus macaques, we experimentally confirmed high levels of constitutive TRIM22 expression in the lung. In contrast, TRIM22 expression in many widely used transformed cell lines could only be observed following immune stimulation. Endogenous levels of TRIM22 in non-transformed cells were sufficient to restrict human and avian influenza A virus (IAV) infection by inhibiting the onset of viral transcription independently of cytokine-mediated innate immune defences. Thus, TRIM22 confers a pre-existing (intrinsic) tissue-specific immune barrier to IAV infection in the respiratory tract. We investigated whether the constitutive expression of TRIM22 was a characteristic shared by other ISGs in human lung tissue. Transcriptomic analysis identified a large group of ISGs and IAV immuno-regulatory host factors that were similarly enriched in the lung relative to other mucosal tissues, but whose expression was downregulated in transformed cell-lines. We identify common networks of immune gene downregulation which correlated with enhanced permissivity of transformed cells to initiate IAV replication. Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-existing immune gene expression in the intrinsic intracellular restriction of IAV; findings highly relevant to the immune regulation of many clinically important respiratory pathogens.\n\nAuthor SummaryThe respiratory tract is a major portal of virus entry for many clinically important viruses, including seasonal and pandemic influenza A virus (IAV). We reasoned that cells within the respiratory tract might differentially express antiviral host factors to protect against the constant challenge of viral infection. We found the broad-acting antiviral protein TRIM22, conventionally regarded as an interferon stimulated gene (ISG) product upregulated in response to virus infection, to be constitutively expressed to high levels in the lung. We found that constitutive expression of TRIM22 restricted the initiation of human and avian IAV infection independently of cytokine-mediated innate immune defences. We identified pre-existing tissue-specific and cell-type dependent patterns of constitutive immune gene expression that strongly correlated with enhanced resistance to IAV replication from the outset of infection. Importantly, we show that these constitutive patterns of immune gene expression are lost or downregulated in many transformed cell lines widely used for respiratory virus research. Our data highlight the importance of pre-existing tissue-specific and cell-type dependent patterns of constitutive antiviral gene expression in the intracellular restriction of respiratory viral pathogens not captured in conventional cell culture model systems of infection.

immunology↗

bioRxiv2019

Maximum entropy models elucidate the contribution of metabolic traits to patterns of community assembly

Aim: Maximum entropy (MaxEnt) models promise a novel approach for understanding community assembly and species abundance patterns. One of these models, the \"Maximum Entropy Theory of Ecology\" (METE) reproduces many observed species abundance patterns, but is based on an aggregated representation of community structure that does not resolve species identity or explicitly represent species-specific functional traits. In this paper, METE is compared to \"Very Entropic Growth\" (VEG), a MaxEnt model with a less aggregated representation of community structure that represents species (more correctly, functional types) in terms of their per capita metabolic rates. We examine the contribution of metabolic traits to the patterns of community assembly predicted by VEG and, through aggregation, compare the results with METE predictions in order to gain insight into the biological factors underlying observed patterns of community assembly. Innovation: We formally compare two MaxEnt-based community models, METE and VEG, that differ as to whether or not they represent species-specific functional traits. We empirically test and compare the metabolic predictions of both models, thereby elucidating the role of metabolic traits in patterns of community assembly. Main Conclusions: Our analysis reveals that a key determinant of community metabolic patterns is the \"density of species\" distribution, defined as the intrinsic number of species with metabolic rates in a given range that are available to a community prior to filtering by environmental constraints. Our analysis suggests that appropriate choice of of the density of species in VEG may lead to more realistic predictions than METE, for which this distribution is not defined, and thus opens up new ways to understanding the link between functional traits and patterns of community assembly.

ecology↗