Research ArticleCORONAVIRUS

Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys

See allHide authors and affiliations

Science Translational Medicine  27 Jan 2021:
Vol. 13, Issue 578, eabe8146
DOI: 10.1126/scitranslmed.abe8146
  • Fig. 1 Viral loads and virus titers in swabs and BALF from African green monkeys.

    Two African green monkeys were inoculated with γ-irradiated SARS-CoV-2 (n = 2). Eight African green monkeys were inoculated with infectious SARS-CoV-2 isolate nCoV-WA1-2020. After inoculation, clinical exams were performed during which nose (A), throat (B), and rectal swabs (C) were collected; (D) bronchoalveolar lavages 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 gRNA (left column) and sgRNA (middle column), and in vitro virus titration was performed to detect infectious virus (right column) in these samples. Amount of gRNA and sgRNA in the inocula (γ-irradiated and infectious) is indicated at time point zero. Teal: animals inoculated with γ-irradiated virus; black: animals inoculated with infectious virus and euthanized at 3 dpi; pink: animals inoculated with infectious virus and euthanized at 10 dpi.

  • Fig. 2 Histological changes are observed in the lungs of African green monkeys inoculated with SARS-CoV-2.

    (A to C) African green monkeys were inoculated with γ-irradiated SARS-CoV-2 (n = 2) and euthanized at 3 dpi; eight animals were inoculated with SARS-CoV-2 isolate nCoV-WA1-2020. (D to F) Four of those were euthanized at 3 dpi. (G to I) The remaining four animals were euthanized at 10 dpi. Histological analysis was performed on lung tissue from all animals. (A) Lungs of animals inoculated with γ-irradiated SARS-CoV-2 were normal at 3 dpi. (B) This was further confirmed at high magnification. (C) No SARS-CoV-2 antigen could be detected in lungs from animals inoculated with γ-irradiated SARS-CoV-2. (D) Mildly thickened septa were observed at 3 dpi in animals inoculated with infectious SARS-CoV-2. (E) Alveolar septa are slightly thickened and more cellular at 3 dpi. (F) Cytoplasmic and membrane-associated viral antigen in pneumocytes at 3 dpi. (G) Discrete foci of interstitial pneumonia are apparent at the periphery of the lung at 10 dpi. (H) Alveolar edema (*), type II pneumocyte hyperplasia (arrowheads), increased alveolar macrophages (arrows), and infiltrating lymphocytes and neutrophils are observed at 10 dpi, as well as proliferative nodules associated with terminal airways resembling obstructive bronchiolitis (OB). (I) Rare viral antigen could be detected in mononuclear cells, presumably alveolar macrophages, with cytoplasmic debris (arrows) at 10 dpi; background blush is observed in alveolar proteinaceous fluid (*), but pneumocytes do not exhibit immunoreactivity (arrowheads). Magnification, ×20 (scale bars: 1 mm) (A to C) and ×400 (scale bars: 0.05 mm) (D to I).

  • Fig. 3 Single-cell sequencing reveals viral dynamics in lung tissue.

    (A) UMAP projection of scRNA-seq 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. NK, natural killer. (B) Validation of cell type identities using marker gene sets. The intensity of the purple color represents higher expression of the indicated marker set. Gray coloring indicates that the cell did not express any genes in the marker set. (C) Viral load in cells isolated from the lungs was evaluated via qRT-PCR for gRNA and grouped for all lobes across each animal in the indicated groups. (D) The percentage of cells identified by scRNA-seq that were positive for any reads aligning to the viral genome by dpi is reported. (E) Percentage of cells from the 3-dpi samples positive for any reads aligning to the viral genome grouped by cell type. DC, dendritic cell. (F) The number of cells grouped by cell type with reads aligning to other locations across the viral genome, all normalized to the number of cells expressing nucleocapsid (N) gene. M, membrane; E, envelope; S, spike. Genes are ordered from the 3′ to 5′ end of the SARS-CoV-2 genome. (G) ISH for viral spike RNA in lung tissues at 3 dpi. Viral RNA staining is shown in red at ×100 magnification (scale bar: 0.2 mm) and ×400 magnification (scale bar: 0.05 mm).

  • Fig. 4 Macrophage populations in the lungs are dynamic during SARS-CoV-2 infection.

    (A) Graphs depicting PC analysis of lung macrophages. The x axis is PC1, and the y axis is PC2. Experimental groups are plotted independently. Each point is an individual cell and is colored on the basis of the expression of MARCO. The lines on the PC graphs are for reference across the samples and represent matching locations. (B) Quantification of the percentage of macrophages that are MARCO (purple) or MARCO+ (green) across the three different experimental groups. (C) The MARCO+ macrophage PC analysis (density plots) was plotted by histograms representing the experimental groups. PC1 is shown on the left, and PC2 is shown on the right. The heatmap below showing the individual cells (columns) sorted based on their location along PC1 or PC2. Top genes showing high correlation along that PC are clustered in rows. A few of the gene names are noted just to the right of the heatmaps. (D) The density plots and histograms are shown as in (C) for MARCO cells. (E and F) Comparison between the individual cell identity (columns) and the cluster identity (rows) based on an unbiased identification algorithm at 3 dpi (E) or 10 dpi (F) for SARS-CoV-2−infected animals. The color intensity represents the percent of individual cells in the cluster that match the identified phenotype.

  • Fig. 5 Single-cell sequencing of mediastinal lymph nodes shows resolution of inflammatory response.

    (A) UMAP projection of single-cell sequencing data from cells isolated from the mediastinal lymph nodes of all 10 animals combined. Each point represents an individual cell, and cells are colored on the basis of their cell type. The names of the cell types are placed next to their largest cluster. (B) Single gene expression analysis was used to validate cell type identifications. (C) Percent of total gene counts for each cell for a subset of interferon-responsive genes (y axis). The x axis denotes cell types and experimental group. (D) Percentage of each cell population (x axis) that is actively dividing (stage G2-M or S) as determined by a profile of gene expression. Each point is an individual animal, and bars represent the mean and SD of the samples. (E) The percentage of plasma cells in each sample compared relative to the total cell number is plotted. (F) The percentage of plasmablast cells relative to the number of B cells is plotted. *P < 0.05, one-way ANOVA.

  • Table 1 Clinical signs observed in African green monkeys inoculated with irradiated or infectious SARS-CoV-2.

    Animals were observed daily according to a standardized scoring sheet (19); the same person assessed the animals throughout the study. N/A, not available.

    InoculumAnimalClinical signs observed
    1 to 3 dpi
    Clinical signs observed
    4 to 10 dpi
    Observations at necropsy
    Irradiated SARS-CoV-2AGM1Reduced appetite.N/ANone.
    Euthanized 3 dpi.
    AGM2Reduced appetite.N/ANone.
    Euthanized 3 dpi.
    SARS-CoV-2AGM3Reduced appetite.N/AGross lung lesions; cervical and
    mediastinal lymph nodes enlarged.
    Euthanized 3 dpi.
    AGM4Tachypnea; reduced appetite.N/AGross lung lesions; mediastinal
    lymph nodes mildly enlarged.
    Euthanized 3 dpi.
    AGM5Tachypnea; reduced appetite.N/AGross lung lesions; mediastinal
    lymph nodes enlarged.
    Euthanized 3 dpi.
    AGM6Reduced appetite.N/AGross lung lesions; mediastinal
    lymph nodes enlarged.
    Euthanized 3 dpi.
    AGM7Reduced appetite.Reduced appetite.Gross lung lesions; mediastinal
    lymph nodes enlarged.
    Recovered at 5 dpi.
    AGM8Hunched posture; tachypnea;
    severely reduced appetite.
    Hunched posture; tachypnea;
    severely reduced appetite.
    Lungs consolidated; mediastinal
    lymph nodes enlarged and
    hemorrhagic.
    Recovered at 9 dpi.
    AGM9Tachypnea; reduced appetite.Tachypnea; reduced appetite.Gross lung lesions; mediastinal
    lymph nodes enlarged.
    Recovered at 6 dpi.
    AGM10Hunched posture; tachypnea;
    coughing; reduced appetite.
    Reduced appetite.None.
    Recovered at 5 dpi.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/scitranslmed.abe8146/DC1

    Fig. S1. Clinical scores, gross lung lesions, and viral loads in respiratory and GI tract of African green monkeys inoculated with SARS-CoV-2.

    Fig. S2. Histological changes in intestinal tract and lymph nodes of African green monkeys infected with SARS-CoV-2.

    Fig. S3. Percentage of cell types in the lung determined by scRNA-seq.

    Fig. S4. Coverage of reads from the pseudo-bulk data across the SARS-CoV-2 genome.

    Fig. S5. Clustering bias across cell populations in the lungs.

    Fig. S6. Gene set enrichment analysis of macrophages across the PCs.

    Fig. S7. Comparison of different cell populations in the mediastinal lymph node.

    Fig. S8. Analysis of T cell populations in lung and mediastinal lymph node.

    Table S1. Virus isolation from tissues of African green monkeys inoculated with SARS-CoV-2 and euthanized at 3 and 10 dpi.

    Data file S1. Raw data.

    Data file S2. Gene correlations along PCs of the macrophages and interferon stimulated gene (ISG) gene set.

  • The PDF file includes:

    • Figure S1. Clinical scores, gross lung lesions and viral loads in respiratory and gastrointestinal tract of African green monkeys inoculated with SARS-CoV-2.
    • Figure S2. Histological changes in intestinal tract and lymph nodes of African green monkeys infected with SARS-CoV-2.
    • Figure S3. Percentage of cell types in the lung determined by single cell RNA sequencing.
    • Figure S4. Coverage of reads from the pseudo-bulk data across the SARS-CoV-2 genome.
    • Figure S5. Clustering bias across cell populations in the lungs.
    • Figure S6. Gene set enrichment analysis of macrophages across the principal components.
    • Figure S7. Comparison of different cell populations in the mediastinal lymph node.
    • Figure S8. Analysis of T cell populations in lung and mediastinal lymph node.
    • Table S1. Virus isolation from tissues of African green monkeys inoculated with SARS-CoV-2 and euthanized at 3 and 10 days post inoculation.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article