Biology subjects

Smyth, R.

Publications and source records attributed to Smyth, R..

9 recordsLinked to original sources

bioRxiv2026

Influenza Virus Infection of an Immunocompetent Organotypic Model of the Human Respiratory Mucosa

Respiratory infectious diseases are among the leading causes of global morbidity and mortality and remain a major public health concern. Progress in understanding early host-pathogen interactions has been hampered by the limited physiological relevance of existing experimental systems. Different models mimicking the human respiratory epithelium have been developed to study viral infections in vitro, such as tridimensional (3D) tissue models and organoids. However, many lack key features of human tissue architecture, particularly the lamina propria or immune cells. To address these limitations, we established an immunocompetent 3D model of the human respiratory mucosa by combining nasal epithelial cells isolated from nasal brushings, fibroblasts from mid-turbinate nasal biopsies, and macrophages derived from blood monocytes. These cells were sequentially seeded into collagen-chitosan scaffolds, resulting in a reconstructed respiratory mucosa closely resembling the in vivo nasal tissues. To further confirm the physiological relevance of the model, we infected it with influenza A virus. The mucosa model supported viral replication in the epithelium and consequently showed increased secretion of inflammatory cytokines and upregulation of type I interferon related genes, enabling the monitoring of early antiviral innate immune responses in a physiologically relevant context.

bioengineering↗

bioRxiv2026

Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral screening in flaviviruses

As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmuehl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.

microbiology↗

bioRxiv2025

Complete post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

Transfer RNAs play a critical role in protein synthesis by matching mRNA codons to their corresponding amino acids. Their post-transcriptional modifications, shaping structure, stability, and codon decoding, are now recognized as key regulators of translation and cellular adaptation, including in bacterial pathogens. Here, we provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including the non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of [m2A]37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and a dedicated pseudouridine synthase responsible for [{Psi}]32 formation. Besides, heterogenous modification patterns were observed in tRNALeu(UAA) and tRNALys(CAA), highlighting a complex interplay in anticodon hypermodification. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of the decoding properties of the reduced S. aureus tRNA set, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework for future research aimed at dissecting the role of specific tRNA modifications in S. aureus physiology and pathogenesis.

molecular biology↗

bioRxiv2025

Neutrophil myeloperoxidase as a functional biomarker for RSV severity: implications for in vitro therapeutic screening.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet effective therapeutics are lacking. The aim of this study is to develop an in vitro model that recapitulates key clinical outcomes in infants with RSV bronchiolitis to help accelerate the discovery of effective therapeutics. Neutrophil activation and influx into the airways are hallmarks of severe RSV infection, but these responses are difficult to quantify in clinical trials. Here we profile peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) and identify myeloperoxidase (MPO) as a key indicator of disease severity when compared to age matched controls. To mechanistically model this response, we established a paediatric airway epithelial air-liquid interface (ALI) system incorporating an endothelial layer and primary neutrophils to recapitulate the tissue microenvironment at the blood-airway barrier. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. We then evaluated two antiviral candidates (remdesivir (RDV) and RSV604) to assess their ability to modulate neutrophil activation. While both compounds reduced viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. These findings establish MPO as both a biomarker of RSV disease severity and a functional readout of therapeutic efficacy and demonstrate that targeting neutrophildriven inflammatory pathways may be critical for reducing pathology in infant RSV infection. Take home messageAntiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/682115v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@170058corg.highwire.dtl.DTLVardef@17c344corg.highwire.dtl.DTLVardef@14d79baorg.highwire.dtl.DTLVardef@172ac3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m6A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N6-methyladenosine (m6A), it remains controversial whether if m6A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m6A epitranscriptome on a single molecule level and elucidate how m6A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m6A methylated, [~]3x more methylated than host mRNAs. Previously unreported m6A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m6A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m6A. Concordantly, pharmacological reduction of viral m6A using the m6A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m6A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m6A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.

microbiology↗

bioRxiv2023

PI4KIII3β inhibition reduces rhinovirus associated cell shedding and ciliary dysfunction

BackgroundPatients with chronic obstructive pulmonary disease (COPD) experience respiratory exacerbations, many of which are associated with rhinoviruses. Current treatment strategies do not target the pathogenic rhinovirus trigger. Research questionWhat is the immediate effect of rhinovirus on the ciliated respiratory epithelium and can viral replication and epithelial toxicity be reduced by targeted PI4KIII{beta} inhibition. MethodsShort (24h) and longer (7 days) rhinovirus infection were explored in primary ciliated airway epithelial cultures from multiple healthy and COPD patients using high-speed video microscopy, viral titration assays and immunofluorescence studies. Ciliated epithelial cultures were pre-treated with a PI4KIII{beta} (GSK533) blocker prior to infection to assess efficacy against rhinovirus. Cytokine and chemokine production were assessed by multiplex immunoassays. ResultsWithin hours of infection rhinovirus co-localised with ciliated cells causing extensive apoptosis-associated shedding of predominantly ciliated cells within 24 hours. Viral replication that peaked at day 1 and cleared by day 7, was associated with dramatic loss of ciliated cells confirmed by reduced ciliary activity and ciliary DNAI2 protein expression. Ciliary beat frequency (CBF) of remaining cilia was significantly reduced by day 7 in cultures from COPD. Infection was partly dependent on PI4KIII{beta} with the GSK533 blocker reducing viral replication while preserving ciliary activity. High levels of pro-inflammatory mediators were secreted by infected cells. ConclusionDecreased ciliation due to rhinovirus infection is likely to impair mucociliary clearance in healthy individuals and COPD patients, contributing to the pathophysiology of COPD exacerbations. PI4KIII{beta} inhibition blocks viral replication, helping to preserve ciliary activity. Take home messageRhinovirus replication in the healthy and COPD respiratory epithelium is mediated by PI4KIII{beta} and intracellular PI4P platform formation. Inhibition of PI4KIII{beta} reduced viral replication and ciliated cell loss.

cell biology↗

bioRxiv2023

Sequencing accuracy and systematic errors in nanopore direct RNA sequencing

Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been exploring and expanding the potential of dRNA-seq, the sequencing accuracy and error patterns remain understudied. We present the first comprehensive evaluation of accuracy and systematic errors in dRNA-seq data from diverse species, as well as synthetic RNA. Deletions significantly outnumbered mismatches/insertions, while the median read accuracy exhibited species-level variation. In addition to homopolymer errors, we observed systematic biases across nucleotides and heteropolymeric motifs in all species. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines/adenines. Moreover, the systematic errors were strongly dependent on local sequence contexts. By examining raw signal data, we identified underlying signal-level features potentially associated with the error patterns. While read quality scores approximated error rates at base and read levels, failure to detect DNA adapters may lead to data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (base-calling) artefacts. Lastly, we discuss the implications of such error patterns for downstream applications of dRNA-seq data.

bioinformatics↗

bioRxiv2021

Spatial and temporal analysis of neutrophil trans-epithelial migration in response to RSV infection

The recruitment of neutrophils to the infected airway occurs early following respiratory syncytial virus (RSV) infection and high numbers of activated neutrophils in airway and blood is associated with the development of severe disease. Here, we investigated whether trans-epithelial migration across primary human airway epithelial cells (AECs) is sufficient and necessary for neutrophil activation during RSV infection. Using flow cytometry, we identified three populations of neutrophils in our in vitro model; those in suspension in basolateral and apical compartments and those that migrated and adhered to AECs. After 1h incubation, the number of adherent neutrophils was significantly greater following RSV infection compared to mock infected. We found that, when migration occurred, neutrophil expression of CD11b, CD62L, CD64, NE and MPO increased in all compartments. However, this did not occur when neutrophils were prevented from migrating. This suggests that the heightened neutrophil activation we detected in the basolateral compartment may be due to reverse migrating neutrophils, as has been suggested by clinical observations. Using live-cell fluorescent microscopy, we then profiled the early temporal and spatial movement and adherence of human neutrophils during migration. Our findings suggest three main phases of early neutrophil recruitment and behaviour in the airways during RSV infection, with neutrophil recruitment, activation and adherence to RSV infected AECs, with clustering, occurring within the first 20 minutes. This work and the model we developed could provide new insight into how neutrophil activation and a dysregulated neutrophil response to RSV mediates disease severity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/463016v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16639c5org.highwire.dtl.DTLVardef@5aeeacorg.highwire.dtl.DTLVardef@1eb96ceorg.highwire.dtl.DTLVardef@1f9b899_HPS_FORMAT_FIGEXP M_FIG Building on previous work of neutrophil function we propose 3 main phases of early neutrophil recruitment and behaviour in the airways during RSV infection. Phase 1. Initial chemotaxis and adherence: Here unstimulated circulating neutrophils expressing baseline levels of CD11b migrate across infected AECs in response to chemotactic signals in the apical supernatant. Some neutrophils remain adherent to the infected AECs. Phase 2: Activation and reverse migration: once on the apical side of the epithelium, neutrophils increase expression of CD11b and other activation associated markers, and some activated neutrophils undergo reverse migration. Neutrophils with greater expression of CD11b are detected on the basolateral side Phase 3: Amplified chemotaxis and clustering: after 20 minutes, adherent neutrophils begin to rapidly cluster on RSV infected primary airway epithelial cells cultures, mediated by signalling from a dying neutrophil. Drawing created using BioRender.com. C_FIG

immunology↗

bioRxiv2019

Neutrophil: Airway Epithelial Interactions Result in Increased Epithelial Damage and Viral Clearance during RSV Infection

Respiratory syncytial virus (RSV) is a major cause of paediatric respiratory disease. Large numbers of neutrophils are recruited into the airways of children with severe RSV disease. It is not clear whether or how neutrophils enhance recovery from disease or contribute to its pathology. Using an in vitro model of the differentiated airway epithelium, we found that addition of physiological concentrations of neutrophils to RSV infected nasal cultures was associated with greater epithelial damage with lower ciliary activity, cilia loss, less tight junction expression (ZO-1) and more detachment of epithelial cells than seen with RSV infection alone. This was also associated with a decrease in infectious virus and fewer RSV positive cells in cultures after neutrophil exposure compared to pre-exposure. Epithelial damage in response to RSV infection was associated with neutrophil activation (within 1h), and neutrophil degranulation with significantly greater cellular expression of CD11b, MPO and higher neutrophil elastase and myeloperoxidase activity in apical surface medias compared to that from mock-infected AECs. We also recovered more apoptotic neutrophils from RSV infected cultures (>40%), compared to <5% in mock infected cultures after 4h. The results of this study could provide important insights into the role of neutrophils in host response in the airway.

immunology↗