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 forin vitroandin vivoSARS-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.

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15–35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, withGzma^-/-mouse studies having informed our understanding of GZMA’s physiological function. We show herein thatGzma^-/-mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereasNntis truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated inGzma^-/-mice; however, the presence ofNntand the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6JGzma^S211Amouse provided the first insights into GZMA’s 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 ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain hadNntsequencing reads inconsistent with a C57BL/6J genetic background.Nntand C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

BACKGROUND How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. The K18-hACE2 mouse model has been widely used for evaluation of new interventions for COVID-19. METHOD Herein we use RNA-Seq data and bioinformatics approaches to compare the transcriptional responses in the SARS-CoV-2 infected lungs of K18-hACE2 mice with those seen in humans. RESULTS Overlap in differentially expressed genes was generally poor (≈20-30%), even when multiple studies were combined. The overlap was not substantially improved when a second mouse model was examined wherein hACE was expressed from the mouse ACE2 promoter. In contrast, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between the species. CONCLUSION As immunity and immunopathology are the focus of most studies, these hACE2 transgenic mouse models can thus be viewed as representative and relevant models of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which can result in severe disease, often characterised by a ‘cytokine storm’ and the associated acute respiratory distress syndrome. However, many infections with SARS-CoV-2 are mild or asymptomatic throughout the course of infection. Although blood biomarkers of severe disease are well studied, less well understood are the inflammatory signatures in lung tissues associated with mild disease or silent infections, wherein infection and inflammation are rapidly resolved leading to sequelae-free recovery. Herein we described RNA-Seq and histological analyses of lungs over time in an omicron BA.1/K18-hACE2 mouse infection model, which displays these latter features. Although robust infection was evident at 2 days post infection (dpi), viral RNA was largely cleared by 10 dpi. Acute inflammatory signatures showed a slightly different pattern of cytokine signatures compared with severe infection models, and where much diminished 30 dpi and absent by 66 dpi. Cellular deconvolution identified significantly increased abundance scores for a number of anti-inflammatory pro-resolution cell types at 5/10 dpi. These included type II innate lymphoid cells, T regulatory cells, and interstitial macrophages. Genes whose expression trended downwards over 2 – 66 dpi included biomarkers of severe disease and were associated with ‘cytokine storm’ pathways. Genes whose expression trended upward during this period were associated with recovery of ciliated cells, AT2 to AT1 transition, reticular fibroblasts and innate lymphoid cells, indicating a return to homeostasis. Very few differentially expressed host genes were identified at 66 dpi, suggesting near complete recovery. The parallels between mild or subclinical infections in humans and those observed in this BA.1/K18-hACE2 mouse model are discussed with reference to the concept of “protective inflammation”.