Biology subjects

Rawle, D. J.

Publications and source records attributed to Rawle, D. J..

4 recordsLinked to original sources

bioRxiv2021

An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmAs physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmAs bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that {approx}27% of Run Accessions and {approx}38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

immunology↗

bioRxiv2021

SARS-CoV-2 infection models using in vivo and in vitro hACE2-lentivirus transduction

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development. AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

microbiology↗

bioRxiv2020

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19 patients. The initial step to identifying potential candidates usually involves in vitro screening. Here we describe a simple rapid bioassay for drug screening using Vero E6 cells and inhibition of cytopathic effects (CPE) measured using crystal violet staining. The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. A key refinement involves a simple growth assay to identify drug concentrations that cause cellular stress or "cytomorbidity", as distinct from cytotoxicity or loss of viability. For instance, hydroxychloroquine shows anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities.

microbiology↗

bioRxiv2020

Systems vaccinology analysis of a recombinant vaccinia-based vector reveals diverse innate immune signatures at the injection site

Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFN{gamma}, TNF and IL1{beta}, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.

systems biology↗