SARS-CoV-2 infection dynamics in lungs of African green monkeys

Abstract

Detailed knowledge about the dynamics of SARS-CoV-2 infection is important for unraveling the viral and host factors that contribute to COVID-19 pathogenesis. Old-World nonhuman primates recapitulate mild-moderate COVID-19 cases, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with SARS-CoV-2 or inactivated virus to study the dynamics of virus replication throughout the respiratory tract. RNA sequencing of single cells from the lungs and mediastinal lymph nodes allowed a high-resolution analysis of virus replication and host responses over time. Viral replication was mainly localized to the lower respiratory tract, with evidence of replication in the pneumocytes. Macrophages were found to play a role in initiating a pro-inflammatory state in the lungs, while also interacting with infected pneumocytes. Our dataset provides a detailed view of changes in host and virus replication dynamics over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

Figure 1.. Viral loads and virus titers in swabs and bronchoalveolar lavage fluid.

Two African green monkeys (AGM) were inoculated with gamma-irradiated SARS-CoV-2 (n=2). Eight AGM were inoculated with infectious SARS-CoV-2 isolate nCoV-WA1–2020. After inoculation, clinical exams were performed during which nose, throat and rectal swabs were collected; bronchoalveolar lavages (BAL) were performed at 1, 3, and 5 dpi on the four animals remaining in the study through 10 dpi and viral loads and titers were measured. qRT-PCR was performed to detect genomic (left column) and subgenomic RNA (middle column), and in vitro virus titration was performed to detect levels of infectious virus (right column) in these samples. Amount of gRNA and sgRNA in the inocula (gamma-irradiated and infectious) is indicated at timepoint zero. Teal: animals inoculated with gamma-irradiated virus; black: animals inoculated with infectious virus and euthanized at 3 dpi; pink: animals inoculated with infectious virus and euthanized at 10 dpi. Where indicated, identical symbols have been used to identify individual animals throughout the figures in this manuscript.

Figure 2.. Histological changes in the lungs of African green monkeys inoculated with SARS-CoV-2.

African green monkeys were inoculated with gamma-irradiated SARS-CoV-2 (n=2) and euthanized at 3 dpi; eight animals were inoculated with SARS-CoV-2 isolate nCoV-WA1–2020 and four of those were euthanized at 3 dpi, and four at 10 dpi. Histological analysis was performed on lung tissue from all animals. (a) Lungs of animals inoculated with gamma-irradiated SARS-CoV-2 were normal at 3 dpi. (b) Mildly thickened septa were observed at 3 dpi in animals inoculated with infectious SARS-CoV-2. (c) Discrete focus of interstitial pneumonia at the periphery of the lung at 10 dpi. (d) High magnification of normal lung tissue in (a). (e) Alveolar septa are slightly thickened and more cellular at 3 dpi. (f) Alveolar edema (*), type II pneumocyte hyperplasia (arrowhead), increased alveolar macrophages (arrow) and infiltrating lymphocytes and neutrophils are observed at 10 dpi, as well as proliferative nodules associated with terminal airways resembling obstructive bronchiolitis (OB). (g) No SARS-CoV-2 antigen could be detected in lungs from animals inoculated with gamma-irradiated SARS-CoV-2. (h) Cytoplasmic and membrane-associated viral antigen in pneumocytes at 3 dpi. (i) Rare viral antigen could be detected in mononuclear cells, presumably alveolar macrophages, with cytoplasmic debris (arrow) at 10 dpi; background blush is observed in alveolar proteinaceous fluid (*), but pneumocytes do not exhibit immunoreactivity (arrowhead). Magnification a-c 40x, d-i 400x.

Figure 3.. Single cell sequencing and viral dynamics in lung tissue.

A. UMAP projection of single-cell RNA sequencing data from whole lung sections from all 10 animals combined. Each point is an individual cell; colors are based on cell type annotation. Cell names are shown next to their largest cluster. B. Validation of cell type identities using marker gene sets. The darker the purple in each cell the higher its expression of the marker set. Grey means the cell did not express any genes in the marker set. C. Viral load information from the lungs via qRT-PCR for gRNA, grouped for all lobes across all animals. D. Percentage of cells identified by single cell RNA-sequencing that are positive for any reads aligning to the viral genome by days post inoculation. E. Percentage of cells from the 3 dpi samples positive for any reads aligning to the viral genome grouped by cell type. F. The number of cells grouped by cell type (colored to match the UMAP in A) with reads aligning to various other locations across the viral genome, all normalized to the number of cells expressing N. Genes are ordered from the 3’ to 5’ end of the SARS-CoV-2 genome. G. ISH for viral S RNA in lung tissues at 3 dpi (viral RNA stains red) at 100x magnification and 400x magnification.

Figure 4.. Changes in macrophage populations in the lungs during SARS-CoV-2 infection.

A. Principal component analysis of the macrophages. The x-axis is pc1 and the y-axis is pc2. The graph is split by the experimental groups. Each point is an individual cell and is colored based on the expression of MARCO with grey representing zero expression and the dark purple represent increased expression. The lines on the PC graphs are for reference across the samples and represent matching locations. B. The percentage of macrophages that are MARCO + (green) or MARCO − (purple) across the three different conditions (x-axis) C. The MARCO+ macrophages principal component analysis (density plots) grouped by the different conditions. Blue: animals inoculated with gamma-irradiated virus and euthanized on 3 dpi; grey: animals inoculated with SARS-CoV-2 and euthanized at 3 dpi; pink: animals inoculated with SARS-CoV-2 and euthanized at 10 dpi. The left density plot is pc1 with the heatmap below showing the individual cells (columns) sorted based on their location along pc1 with top genes showing high correlation along that principal component clustered (rows). Darker blue means low to no expression and a dark red color means high expression. A similar graph, but for PC2 is on the right. A few of the gene names are noted just to the right of the heatmaps. D. A similar set of graphs as in panel C but for the MARCO− cells. E. Showing the comparison between the individual cell identity (columns) and the cluster level identity (rows) based on the unbiased identification algorithm at 3 dpi for SARS-CoV-2-infected animals. The colors represent the % of individual cells in the cluster that match the identified phenotype. White means no individual cell in that cluster match the phenotype; whereas the darker the blue the higher the percentage of matching cells. F. A similar graph to E at 10 dpi for SARS-CoV-2-infected animals.

Figure 5.. Single cell sequencing of mediastinal lymph nodes.

A. UMAP projection of single cell sequencing data from lymph nodes of all 10 animals combined. Each point represents an individual cell and cells are colored based on their cell type. The names of the cell types are placed next to their largest cluster. B. Single gene expression to validate cell type identifications in A. For each graph in this panel, grey means the cell had zero expression and the darker the purple, the higher the expression of that gene. C. percent of total gene counts for each cell for a subset of interferon responsive genes (y-axis). The x-axis is split by the cell types and further divided by sample: blue represents animals inoculated with gamma-irradiated virus and euthanized at 3 dpi; grey represents animals inoculated with SARS-CoV-2 and euthanized at 3 dpi; pink represents animals inoculated with SARS-CoV-2 and euthanized at 10 dpi. Individual cells are removed for clarity and only densities of cells across the populations are shown in violin plots. D. Percentage of each cell population (x-axis) that is actively dividing (stage G2M or S) as determined by a profile of gene expression. Each point is an individual animal and bars represent the mean and standard deviation of the samples. E. Percentage of plasma cells in each animal’s sample compared it’s total cell numbers. F. Percentage of plasmablast cells in each animal’s sample compared to the total B cell population.