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      ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

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          Abstract

          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. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR -/- and IL-28RA -/- mouse 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 summary

          SARS-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.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Gene Ontology: tool for the unification of biology

            Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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              The Sequence Alignment/Map format and SAMtools

              Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Data curationRole: Formal analysisRole: Visualization
                Role: Investigation
                Role: Investigation
                Role: Formal analysis
                Role: InvestigationRole: ResourcesRole: Visualization
                Role: InvestigationRole: ResourcesRole: Visualization
                Role: InvestigationRole: Resources
                Role: Visualization
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                2 July 2021
                July 2021
                : 17
                : 7
                : e1009723
                Affiliations
                [1 ] Immunology Department, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
                [2 ] School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland, Australia
                [3 ] Australian Infectious Disease Research Centre, GVN Center of Excellence, Brisbane, Queensland, Australia
                Icahn School of Medicine at Mount Sinai, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0001-7248-0101
                https://orcid.org/0000-0001-9134-0104
                https://orcid.org/0000-0002-5789-4928
                https://orcid.org/0000-0001-5957-2853
                https://orcid.org/0000-0003-3205-4970
                https://orcid.org/0000-0002-5710-9942
                https://orcid.org/0000-0001-8986-9025
                Article
                PPATHOGENS-D-21-00524
                10.1371/journal.ppat.1009723
                8282004
                34214142
                18fe8c8c-d4b9-4fc8-9268-451c7a6d8c55
                © 2021 Rawle et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 March 2021
                : 17 June 2021
                Page count
                Figures: 7, Tables: 0, Pages: 37
                Funding
                Funded by: Clive Berghofer and Brazil Family Foundation
                Award ID: Philanthropic
                Award Recipient :
                Funded by: clive berghofer and the brazil family foundation
                Award ID: Philanthropic
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000925, National Health and Medical Research Council;
                Award ID: APP1173880
                Award Recipient :
                Funded by: australian infectious diseases research centre
                Award ID: intramural seed grant
                We thank Clive Berghofer and the Brazil Family Foundation (and many others) for their generous philanthropic donations to support SARS-CoV-2 research at QIMR Berghofer MRI ( https://www.qimrberghofer.edu.au/) (intramural grants awarded to A.S. and D.J.R). The project was also partly funded by an intramural seed grant from the Australian Infectious Diseases Research Centre ( https://aidrc.org.au/) awarded to A.S. A.S. holds an Investigator grant from the National Health and Medical Research Council (NHMRC, https://www.nhmrc.gov.au/) of Australia (APP1173880). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Custom metadata
                vor-update-to-uncorrected-proof
                2021-07-15
                All raw sequencing data (fastq files) are available from the Sequence Read Archive (SRA), BioProject accession: PRJNA701678. All other data is available within the paper and supporting information files.

                Infectious disease & Microbiology
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