Bat organoids reveal antiviral responses at epithelial surfaces

Abstract

Bats can host viruses of pandemic concern without developing disease. The mechanisms underlying their exceptional resilience to viral infections are largely unresolved, necessitating the development of physiologically relevant and genetically tractable research models. Here, we developed respiratory and intestinal organoids that recapitulated the cellular diversity of the in vivo epithelium present in Rousettus aegyptiacus, the natural reservoir for the highly pathogenic Marburg virus (MARV). In contrast to human counterparts, bat organoids and mucosal tissue exhibited elevated constitutive expression of innate immune effectors, including type I interferon-ε (IFNε) and IFN-stimulated genes (ISGs). Upon infection with diverse zoonotic viruses, including MARV, bat organoids strongly induced type I and III IFN responses, which conferred robust antiviral protection. Type III IFNλ3 additionally displayed virus-independent self-amplification, acting as an ISG to enhance antiviral immunity. Our organoid platform reveals key features of bat epithelial antiviral immunity that may inform therapeutic strategies for viral disease resilience.

Fig. 1. R. aegyptiacus nasal, bronchial and alveolar airway organoids contain diverse cell types.

a, Uniform manifold approximation and projection (UMAP) of EC types of lung and tracheal tissue, identified using scRNA-seq from a captive-bred R. aegyptiacus. Clusters labeled with ‘/’ indicate mixed identities: AT1/basal (AT1 lung or basal cells from the lung or trachea); AT1/suprabasal (AT1 lung or suprabasal trachea); secretory/club (goblet or club cells); ciliated/mixed (cells with ambiguous identity expressing a ciliated marker). b, Representative KRT5 or SFTPC immunofluorescence staining (top) and 4′,6-diamidino-2-phenylindole (DAPI) nucleus counterstaining (bottom) in R. aegyptiacus lung sections (n = 3). Arrowheads indicate KRT5⁺ basal or SFTPC⁺ AT2 cells. Scale bars, 50 µm. c, Schematic of the main adult respiratory epithelial stem cell types: KRT5⁺TP63⁺ basal cells in the upper and lower conducting airways and SFTPC⁺SFTPB⁺ AT2 cells in the alveolar epithelium. d, Representative KRT5 and acetylated tubulin immunofluorescence staining in differentiated nasal^ORG (left) and nasal^ALI (right) cultures derived from R. aegyptiacus. Scale bars, 50 µm (left) or 150 µm (right). e, Representative KRT5 (left) or SFTPC (right) immunofluorescence staining in lung^MIX-ORG derived from R. aegyptiacus and grown in complete alveolar medium (Methods). The arrowhead highlights SFTPC⁺ cells. Scale bars, 50 µm. f, Representative SFTPC immunofluorescence staining in alv^ORG derived from R. aegyptiacus. Scale bar, 100 µm. g, UMAP of the R. aegyptiacus-derived alv^ORG scRNA-seq dataset showing the cell-type clusters. h, UMAP of the integrated R. aegyptiacus-derived nasal^ALI and bronchial^ALI scRNA-seq dataset showing the cell-type clusters. i, Seurat DotPlot showing the average expression of the markers for each cell cluster in a merged dataset of R. aegyptiacus-derived nasal^ALI + bronchial^ALI and alv^ORG. The dot size represents the percentage of a cell type expressing a given marker. Color intensity represents the average expression value. Dot size was set to a maximum percentage of 50%. (Genes expressed by more than 50% of cells have the same dot size.) The box highlights the low-to-absent expression of the club and goblet cell markers MUC5AC and MUC5B. j, RT–qPCR analysis of FOXJ1, MUC5AC or SCGB1A1 expression (n = 3 each; normalized to EEF1A1, 2^−ΔCt) in R. aegyptiacus-derived nasal^ALI treated with 10 ng ml⁻¹ recombinant human IL-13 or PBS from days 10 to 25 of the ALI culture. k, Representative immunofluorescence staining of MUC5AC and E-cadherin in sections from R. aegyptiacus-derived nasal^ALI treated with IL-13 or PBS as in i. Scale bars, 150 µm. Representative images in d–f,k were derived from n = 3. DAPI was used as a nuclear counterstain in immunofluorescence imaging.

Fig. 2. Microfold cells endogenously arise in bat bronchial organoid-derived ALI cultures.

a, UMAP of the integrated R. aegyptiacus-derived nasal^ALI and bronchial^ALI scRNA-seq dataset showing the ciliated, and mixed ciliated and microfold, cell clusters. b, Seurat VlnPlot analysis of the microfold cell markers SOX8, SPIB, GP2, TNFAPI2, TNFRSF11A, CCL20, TNFAPI2 and AIF1, and the ciliated cell marker FOXJ1 in ciliated cell, mixed ciliated and microfold cells, and GP2⁺ microfold cells of R. aegyptiacus-derived bronchial^ALI. The expression distribution was derived from individual cells. c, Seurat DotPlot showing the average expression of receptor–ligand pair markers for the RANK–RANKL signaling axis (TNFSF11, TNFRSF11A and TNFRSF11B) for clusters 1–10 in the R. aegyptiacus-derived bronchial^ALI scRNA-seq dataset. The dot size represents the percentage of an individual cell type expressing a given marker. Color intensity represents the average expression value. Dot size was set to a maximum percentage of 50%. (Genes expressed by more than 50% of cells have the same dot size.)

Fig. 3. R. aegyptiacus SI organoids recapitulate the cellular diversity of the native bat intestinal epithelium.

a, Representative bright-field image of R. aegyptiacus-derived SI^ORG (n = 3). b, UMAP of the integrated R. aegyptiacus-derived SI^T, SI^ORG and differentiated SI^ORG-DIFF showing the individual cell types. c, Stacked bar plot showing the relative cell-type proportions of cell types in R. aegyptiacus-derived SI^T, SI^ORG and differentiated SI^ORG-DIFF. d, Immunofluoresence of DAPI nucleus counterstaining (left) and AVIL antibody staining (right) in SI^ORG from R. aegyptiacus. The arrow points to an individual AVIL⁺ tuft cell. e, Seurat DotPlot showing the scaled average expression of markers enriched in tuft and brush cells for tuft/brush, and non-tuft/brush, ECs in the R. aegyptiacus-derived nasal^ALI, bronchial^ALI, tracheal^T, lung^T, SI^ORG and SI^T scRNA-seq dataset. The dot size represents the percentage of an individual cell type expressing a given marker. Color intensity represents the average expression value. Dot size was set to a maximum percentage of 50%. (Genes expressed by more than 50% of cells have the same dot size.) f, UMAP of EEC subtypes from the integrated R. aegyptiacus SI^T + SI^ORG (left), SI^ORG (middle) or SI^T (right). Cells with an EEC sublineage are color-coded. g, Seurat FeaturePlot showing the average expression of EEC (CHGA) and EEC sublineage marker genes in individual cells of SI^ORG (top) or SI^T (bottom). ARX, differentiated G/I/L/M/X/D EECs; TPH1, enterochromaffin cells; MLN, M cells; PAX4, EEC progenitor cells; SCT, SCT⁺ S-like cells. The maximum color cutoff for the average expression was set to 3. Cells with an average expression of 3 or greater for a given marker have the same color. Scale bars, 50 µm.

Fig. 4. Bat organoids show heightened expression of innate immune genes, including complement system genes, IFNε and ISGs in comparison to human organoids.

a, Seurat DotPlot showing the average expression of genes associated with the complement system and β-actin (ACTB) in cell types in the R. aegyptiacus (bat) SI^ORG (O) or SI^T (T) scRNA-seq dataset (left), human SI^ORG (middle) or from a published human SI^T scRNA-seq dataset. The dot size represents the percentage of an individual cell type expressing a given marker. Color intensity represents the average expression value. Dot size was set to a maximum percentage of 50%. (Genes expressed by more than 50% of cells have the same dot size.) b, Seurat DotPlot showing the average expression of type I IFNα (sum of all annotated IFNA-like genes), IFNω (sum of all annotated IFNW-like genes), IFNβ (IFNB1), IFNε (IFNE), type II IFNγ (IFNG) and type III IFNλ (sum of all annotated IFNL-like genes) in the R. aegyptiacus (left) and human (right) nasal^ALI (N), bronchial^ALI (B), alv^ORG (A) or SI^ORG (SI) scRNA-seq dataset. Dot size represents the percentage of an individual cell type expressing a given marker. Color intensity represents the average expression value. Dot size was set to a maximum percentage of 50%. c, IFNE mRNA expression (in transcripts per million (TPM) + 0.1, pseudocount) in published bulk RNA data of R. aegyptiacus salivary gland (SG), SI, large intestine (LI), lung, kidney, liver and PBMCs. Each dot represents an individual bat (n = 11–17). Samples from the SI are highlighted in red. d, Representative immunofluorescence of IFNε or isotype control staining (top) and DAPI nucleus counterstaining (bottom) in R. aegyptiacus SI or liver sections (n = 3). Scale bars, 100 µm.

Fig. 5. MARV infection in bat organoids triggers an IFN response.

a, RT–qPCR analysis of intracellular MARV-L expression (normalized to EEF1A1, 2^-ΔCt) in bat SI^ORG(n = 3), alv^ORG(n = 3), nasal^ALI (n = 3) or human bronchial^ALI (n = 3) infected with MARV (Musoke strain) at an estimated multiplicity of infection (MOI) of 0.5–1 (50,000 plaque forming units (PFU) for bat SI^ORG and alv^ORG; 100,000 PFU for bat nasal^ALI or human bronchial^ALI) from day 1 to day 7 (D1–D7) after infection. A two-sided Student’s t-test was used to compare the average intracellular MARV-L mRNA level at each time point to the first analyzed time point (D1) for each condition. P > 0.05, no significant difference, *P < 0.05, **P < 0.01,, ****P < 0.0001. b, EdgeR differential gene expression analysis of bat alv^ORG (n = 3) infected with MARV for 72 h compared to mock-infected (left) or bat nasal^ALI (n = 3) at day 5 after infection compared to day 1 after infection with MARV (right). Each dot represents the expression (in log₂-transformed counts per million (CPM)) and log₂-transformed fold change of a differentially expressed gene (DEG) between two comparisons. Selected ISGs are highlighted in red and labeled. c, Human bronchial^ALI (n = 3) at day 5 after infection compared to day 1 after infection with MARV (left) or 72 h after infection with SeV compared to mock infection (right). Each dot represents the expression (in log₂(CPM)) and log₂(fold change) of a DEG between two comparisons. Selected genes are labeled and highlighted in red for ISGs. d, Heatmap showing normalized gene expression (log₂(CPM + 1)) from bulk RNA-seq for ISGs in human bronchial^ALI (left), bat nasal^ALI (middle) or bat alv^ORG (right) from day 1 to day 7 (D1–D7) after infection with MARV or day 3 in mock infection (M). Each mRNA value represents the average of three biological replicates. e, Heatmap displaying log₂-normalized gene expression of ISGs from reanalyzed public nCounter NanoString data of skin samples (inoculation site) from R. aegyptiacus (bat skin^INOC, n = 4–6) from day 1 to day 28 (D1–D28) after infection with MARV or day 13 in mock infection (D13-M). Each mRNA value represents the average of 4–6 bats in the published dataset.

Fig. 6. Type III IFNs are predominantly induced in bat organoids upon zoonotic virus infection.

a, RT–qPCR analysis of type I IFNB1, type III IFNL1-like (LOC107521777), IFNL3-like (LOC107521776), IFIT1 (LOC107501624) and CCL5 (normalized to EEF1A1, 2^-ΔCt) in virus-infected (SeV, H1N1, VSV-eGFP or MERS-CoV), poly(I:C)-transfected or mock-treated bat nasal^ORG, SI^ORG or alv^ORG. Each mRNA value represents the average of three biological replicates. SeV (S), H1N1 (I) and VSV (V) infections were performed at an MOI of 0.5 for 16 h; poly(I:C) (R) was transfected at 1 µg ml⁻¹ for 16 h and MERS-CoV infections were conducted for 24 (C1), 48 (C2) or 72 h (C3) or mock (MC). b, Heatmap showing normalized gene expression (log₂(CPM + 1)) from bulk RNA-seq in SeV-infected bat SI^ORG (left), SeV-infected bat alv^ORG (middle) or MERS-CoV-infected bat alv^ORG (right) for IFNA-like (sum of all IFNA-like genes), IFNW-like (sum of all IFNW-like genes), IFNK, IFNB1, IFNG, IFNL-like (sum of all IFNL-like genes) (top) and ISGs or the epithelial marker EPCAM (bottom) at D1–D3 after infection. Each mRNA value represents the average of three biological replicates. The single asterisk indicates IFN or ISGs expressed in at least two of three biological replicates. c, Normalized gene expression (CPM + 1) from bulk RNA-seq for IFNE and the ISGs IRF7, MX2 or RTP4 in SeV-infected or mock-infected bat SI^ORG (n = 3) or bat alv^ORG (n = 3). Biological replicates are represented by the dots, with the bar height indicating the average expression. A two-sided Student’s t-test was used to compare the average mRNA expression levels of each gene in SeV-infected organoids versus uninfected organoids at day 3 after infection. NS, not significant; ***P < 0.001, ****P < 0.0001.

Fig. 7. Bat type III IFNλ drive self-amplified antiviral responses in bat organoids.

a, Scatter plots showing the log₂-transformed fold changes in gene expression from bulk RNA-seq comparing nasal^ORG (left), alv^ORG (middle) or SI^ORG (right) treated with universal IFNα2 (x axis) or IFNλ1-like (y axis) to mock-treated controls after 8 h. Individual ISGs are highlighted. IFNL3-like (LOC107521776) is highlighted in red. b, RT–qPCR analysis of IFNL3-like (LOC107521776) in bat SI^ORG or alv^ORG; IFNL3 in human SI^ORG) normalized to EEF1A1 (2^−ΔCt) after treatment with bat IFNλ1-like (bat organoids) or human IFNλ1 (human SI^ORG) for 4 and 8 h. A two-sided Student’s t-test was used to compare IFNL3-like mRNA levels at 4 or 8 h after treatment to mock-treated controls. c, Scatter plot showing the log₂-transformed fold changes in gene expression from bulk RNA-seq comparing SI^ORG treated with universal IFNα2 (x axis) or IFNλ3-like (derived from LOC107521776) (y axis) to mock-treated controls after 8 h. Individual ISGs are highlighted in red. d, RT–qPCR analysis of IFNL3-like (LOC107521776) or MX1 (normalized to EEF1A1, 2^−ΔCt) in bat SI^ORG engineered with Cas9 and IRF9 targeting (sgIRF9) or control (sgScrambled) RNA, treated with or without bat IFNλ1-like for 8 h. A two-sided Student’s t-test was used to compare IFNL3-like mRNA levels between sgIRF9 and sgScrambled SI^ORG at 8 h after treatment. e, As in d but comparing sgIRF9 and sgScrambled SI^ORG after infection with VSV-eGFP at an MOI of 0.05 for 72 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. Type I and type III IFNs protect bat organoids from zoonotic virus infection.

a, Experimental workflow showing the administration of universal IFNα2, IFNλ1-like or IFNλ3-like 12 h before, during or 8 h after treatment with VSV infection of bat nasal^ORG or SI^ORG. Intracellular VSV viral RNA was measured 24 h after infection. b,c, RT–qPCR analysis of intracellular VSV-NP viral RNA in bat SI^ORG (n = 3) (b) or nasal^ORG (n = 3) (c), normalized to EEF1A1 (2^−ΔCt) and expressed as a percentage relative to no IFN control. A two-sided Student’s t-test was used to compare VSV viral RNA levels across before, during and after treatment, and no IFN conditions, comparing each IFN treatment to no IFN control. d, Experimental workflow showing bat nasal^ORG or SI^ORG treated with universal IFNα2 or bat IFNλ1-like for 3 h, followed by washout and incubation in IFN-free medium for 24 h and RNA collection at 0, 3 and 24 h after IFN washout. e,f, RT–qPCR analysis of ISGs (normalized to EEF1A1) in bat SI^ORG (n = 3) (e) or nasal^ORG (n = 3) (f) treated with universal IFNα2 or bat IFNλ1-like at 3 h (left) or 24 h (right) after IFN removal. A two-sided Student’s t-test was used to compare ISG mRNA levels between treatments. g, RT–qPCR analysis of intracellular SINV-eGFP^nsP2-P726G nsP1 RNA (normalized to EEF1A1 (2^−ΔCt) and expressed as fold change relative to the mean of the sgScrambled control) in bat SI^ORG engineered with Cas9 and targeted guide RNA (sgIRF9, sgIFNAR2, sgIFNLR1) or control (sgScrambled), infected with SINV-eGFP^nsP2-P726G. A two-sided Student’s t-test was used to compare SINV nsP1 viral RNA levels between treatment groups. nsP1, nonstructural protein 1. h, RT–qPCR analysis of intracellular MARV-L viral RNA (normalized to EEF1A1) in bat SI^ORG, nasal^ORG or alv^ORG (n = 4) treated with or without IFNs during a 3-day MARV infection. A two-sided Student’s t-test was used to compare MARV viral RNA levels between IFN-treated and untreated controls. i, RT–qPCR analysis of MARV-L viral RNA in bat alv^ORG treated with or without 5 µM ruxolitinib during a 3-day infection. A two-sided Student’s t-test was used to compare MARV viral RNA levels between ruxolitinib and dimethylsulfoxide (DMSO) mock-treated alv^ORG and MARV-infected alv^ORG. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Fig. 1. Establishment and characterization of R. aegyptiacus airway organoids.

a, UMAP of R. aegyptiacus airway cell types resolved by scRNA-seq of lung and trachea tissue from a captive-bred bat. Immune, stromal, and epithelial cell lineages are labeled and color-coded. EC, Endothelial cells, LC, Lymphatic cells, DC, Dendritic cells. b, Seurat DotPlot showing scaled average expression of marker genes for cell clusters in (a), grouped by major tissue-resident lineages. Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). Color intensity represents expression level. c, Seurat DotPlot showing marker expression across epithelial cell types. Dot sizes were set to a maximum percentage of 50%. d, Heatmap showing RT-qPCR analysis of KRT5, SCGB1A1, SFTPC, or SFTPB in mixed lung progenitor organoids (Lung^MIX-ORG) cultured with indicated factors. Expression normalized to EEF1A1 and shown as % of maximum. Each value represents the average relative expression (n = 3). e, Representative immunofluorescence of KRT5 in bat nasal^ORG grown in basal cell organoid medium (EGF, FGF10, Rspondin1, Noggin) (n = 3). Composite image with DAPI nuclei counter stain. Scale, 50 µm. f, Representative brightfield images of nasal^ORG, trachea^ORG, or bronchial^ORG at day four following single-cell passage (n = 3). Scale, 100 µm. g, Representative brightfield (left) and fluorescence image (right) of bat lung^MIX-ORG grown in complete alveolar medium (-Noggin) stained with LysoTracker-Red for 2 hours (n = 3 each). Scale, 100 µm. h, Mean Fluorescence Intensity (MFI) of LysoTracker Red staining in mixed lung^MIX-ORG (n = 3) cultured with indicated factors. Dots, image intensity per single organoid analyzed in Fiji. Two-sided unpaired Mann-Whitney tests were performed to compare MFI distribution of lung^MIX-ORG cultured in different media (**** P < 0.0001). i, Schematic showing alv^ORG derivation from LysoTracker Red⁺ AT2 FACS-sorted cells from lung^MIX-ORG. j, FACS plots showing LysoTracker Red intensity in single gated cells from nasal^ORG or lung^MIX-ORG cultured in complete alveolar medium. Percentages of LysoTracker⁺ cells are shown. k, Representative brightfield image of bat alv^ORG established from FACS-sorted AT2 cells. Scale, 100 µm.

Extended Data Fig. 2. Single-cell RNA sequencing of bat and human airway organoids.

a, UMAP of alv^ORG cell types resolved by scRNA-seq, colored by bat donor (n = 3). b, UMAP of bat nasal^ALI and bronchial^ALI cell types by sample group (left, nasal^ALI or bronchial^ALI) or donor (right, n = 3). c, Seurat VlnPlots showing IRX2 (top) and SIX3 (bottom) expression in bat nasal^ALI, bronchial^ALI, or alv^ORG. Wilcoxon Rank Sum Test used (**** P < 0.0001). d, Seurat DotPlot showing marker expression in alv^ORG clusters. Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). Dot color intensity represents scaled expression level. e, Same as (d) for bat nasal^ALI and bronchial^ALI. f, Seurat VlnPlot showing KNIIFE-cell markers in KRT13⁺ vs. KRT13⁻ cells in the Nasal^ALI scRNA-seq dataset. g, UMAP of human nasal^ALI (top) or bronchial^ALI (bottom) airway cell types resolved by scRNA-seq. Cell types are labeled and color-coded. h, Seurat DotPlot marker gene expression human nasal^ALI (left) or bronchial^ALI (right). Dot sizes were set to a maximum percentage of 50%. Dot color intensity represents scaled expression level. i, Bar plots showing the percentages of rare cell types relative to all cells in the bat nasal^ALI (top) or bronchial^ALI (bottom) scRNA-seq dataset.

Extended Data Fig. 3. Single-cell RNA sequencing of R. aegypticus SI^T and SI^ORG.

a, UMAP of R. aegyptiacus single cell transcriptomes resolved by scRNA-seq of whole SI tissue from a captive-bred bat. Immune, stromal, and epithelial cell lineages are labeled and color-coded. b, UMAP of R. aegyptiacus SI^T cell types in the scRNA-seq dataset. Individual cell types are color-coded and epithelial cell types (hereafter designated as SI^T) are additionally highlighted by text labels. c, Seurat DotPlot showing scaled average expression of marker genes for cell clusters in R. aegyptiacus whole SI tissue scRNA-seq dataset. Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). Dot color intensity represents scaled expression. d, Seurat DotPlot showing the scaled average expression of marker genes for cell clusters in the integrated scRNA-seq data of R. aegyptiacus SI^T epithelial cells (left) and SI^ORG (middle) or differentiated SI^ORG (SI^ORG-DIFF) (right). Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). Dot color intensity represents scaled expression. e, Seurat FeaturePlot showing the average expression of Enteroendocrine cells (EECs expressing CHGA) and EEC sublineage marker genes in individual EECs of SI^ORG (top) or SI^T (bottom). PAX4, EEC progenitor; ARX, differentiated G/I/L/M/X/D EEC; TPH1, Enterochromaffin cells; SCT, SCT⁺, S-like cells; SST, D cells; CCK, I cells; GAST, G cells; GHRL, X cells; MLN, M cells. The maximum expression color cutoff set to 3 (cells with an average expression ≥ 3 of a given marker have the same color).

Extended Data Fig. 4. R. aegyptiacus SI^ORG and SI^T express heightened levels of complement system genes compared to human SI^ORG or human SI^T.

a, Seurat FindMarker differential gene expression analysis comparing bat to human SI^ORG (left), bat to human nasal^ALI (middle) or bat to human bronchial^ALI, cultured side-by-side and analyzed by scRNA-seq. Each dot represents the log₂ transformed sum of Seurat SCT normalized expression gene counts (bat + human) of differentially regulated genes (x axis) and the log₂ fold changes (logFC) from the analysis between the two species (y axis). Positive logFC values indicate upregulated in bat organoids compared to human organoids. Selected complement system genes or interferon stimulated genes (ISGs) are labeled and colored in red. Upregulated markers in human compared to bat organoids are labeled and colored in blue. b, Seurat DotPlot showing the average expression of complement system genes in cell types in the R. aegypticus SI^ORG (O) or SI^T (T) scRNA-seq dataset. Dot size, the percentage of an individual cell type expressing a given marker. Color intensity, the average expression value. Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). c, same as in (b) but in the human SI^ORG scRNA-seq (left) or in a published human SI^T scRNA-seq datasets (ref. ).

Extended Data Fig. 5. Comparison of IFNE or ISG expression in R. aegyptiacus and human organoids, and genetic perturbation of IFNE in bat SI^ORG.

a, Seurat DotPlot showing the average expression of IFNE and cell type-specific marker genes in the integrated bat nasal^ALI + bronchial^ALI (left), or bat SI^ORG scRNA-seq dataset. Dot size, the percentage of an individual cell type expressing a given marker. Color intensity, the average expression value. Dot sizes were set to a maximum percentage of 50% (genes expressed by more than 50% of cells have the same dot size). b, Seurat DotPlot showing the average expression of ISGs in the bat nasal^ALI, bronchial^ALI, alv^ORG or SI^ORG scRNA-seq dataset (left), or human nasal^ALI, bronchial^ALI or SI^ORG scRNA-seq dataset (right). Dot size, the percentage of an individual cell type expressing a given marker. Color intensity, the average expression value. c, Violin plots showing the ISG module enrichment score distribution for conserved ISGs (ref. 33) in scRNA-seq of bat or human nasal^ALI, bronchial^ALI or SI^ORG. The distribution was derived from the enrichment scores of individual cells. Median shown as solid line. A positive score indicates enrichment of ISGs in a culture model. Two-sided Mann-Whitney tests were performed to compare human to bat nasal^ALI, bronchial^ALI or SI^ORG (****: P-value < 0.0001). d, Sanger sequencing trace of PCR amplicons spanning the bat IFNE gene in bat SI^ORG expressing Cas9 and a guide RNA targeting IFNE (sgIFNE) or a non-targeting control guide RNA (sgScrambled). The guide RNA spacer sequence and expected cut site is shown above. e, RT-qPCR analysis of ISGs, normalized to EEF1A1 (2^–ΔCT), in Cas9-sgRNA expressing bat SI^ORG (n = 3). Two-sided unpaired Student’s t-tests were performed to compare ISG expression between sgIFNE-SI^ORG and sgScrambled-SI^ORG (**** P < 0.0001). f, VSV titer measured from the culture supernatant of infected sgIFNE (n = 3) or sgScrambled bat SI^ORG (n = 3) after 8 or 24 hours post infection (hpi). The titer was derived from TCID50 assays performed in VeroE6 cells with five replicates per sample supernatant.

Extended Data Fig. 6. Characterization of virus entry factor expression and antiviral responses to MARV infection in R. aegyptiacus organoids and animals.

a, Seurat DotPlot analysis showing the average expression of virus entry factors of cell types in the bat nasal^ALI, bronchial^ALI, alv^ORG or SI^ORG scRNA-seq dataset. Dot size, the percentage of an individual cell type expressing a given marker. Color intensity, the scaled average expression value. Dot sizes were set to a maximum percentage of 50% (top) or 25% (bottom). Genes expressed by more than 50% or 25% of cells have the same dot size. b, MARV (Musoke) titer (in focus forming units per ml (FFU/ml)) measured from combined solutions of apical washes and basal chamber medium of infected human Bronchial^ALI (n = 3) or bat Nasal^ALI (n = 3) after one-, three- or seven-days post infection. c, Gene ontology (GO) enrichment analysis, performed using clusterProfiler, revealed biological processes significantly enriched among genes upregulated in MARV-infected vs. mock-infected bat alv^ORG, or in bat nasal^ALI at day 5 vs. day 1 post-infection. The top 10 enriched biological processes are shown. The x axis indicates the ratio of enriched to total genes within each GO term. Dot size reflects the number of enriched genes per pathway. d, Heatmap showing normalized gene expression (log₂-counts per million + 1) from bulk RNA-seq for IFN subtypes in MARV-infected or mock-infected bat alv^ORG. Each mRNA value represents the average of three biological replicates. Days post-infection are indicated (D3: day 3 post infection, M: day 3 mock-infected). e, Heatmap displaying log₂-normalized gene expression of MARV NP from reanalyzed public nCounter NanoString data of skin, liver, and colon samples from MARV-infected R. aegyptiacus (n = 4–6). Each mRNA value represents the average of four to six animals present in the dataset. Days post-infection are indicated (D1–D28, D13-M: day 13 mock-infected). f, Heatmap displaying log₂-normalized gene expression of ISGs from reanalyzed public nCounter NanoString data of liver and colon samples from MARV-infected R. aegyptiacus (n = 4–6). Each mRNA value represents the average of four to six animals present in the dataset. Days post-infection are indicated (D1–D28, D13-M: day 13 mock-infected).

Extended Data Fig. 7. Type I/III IFNs drive antiviral gene responses in bat and human organoids.

a, Venn diagrams (generated using eulerr.co) showing the number of significantly upregulated genes determined by bulk RNA-seq in bat nasal^ORG, alv^ORG, or SI^ORG (n = 3 each) following treatment with universal IFNα2 (uIFNα2) or IFNλ1-like compared to mock-treated. The total number of upregulated genes per treatment are indicated. Overlapping regions represent genes commonly induced by both IFNs. b, RT-qPCR analysis of IFIT3 and ISG15 expression, normalized to EEF1A1 (2^–ΔCT), in bat nasal^ORG (n = 3) treated with uIFNα2 or IFNλ1-like for 8 h, with or without ruxolitinib (n = 3). Two-sided unpaired Student’s t-tests were used to compare mRNA levels between ruxolitinib-treated and mock-treated, interferon-stimulated nasal^ORG. (ns: not significant; *** P < 0.001, **** P < 0.0001). c, Gene ontology (GO) enrichment analysis of bulk RNA-seq data, performed with clusterProfiler, revealed biological processes significantly enriched among genes upregulated in uIFNα2 vs. mock-infected bat nasal^ORG, alv^ORG, or SI^ORG after 8 h (n = 3 each). The top 10 enriched biological processes are shown. The x axis indicates the ratio of enriched to total genes within each GO term. Dot size reflects the number of enriched genes per pathway. d, Same as in (c) but for IFNλ1-like vs. mock-treated bat nasal^ORG, alv^ORG, or SI^ORG. e, Scatter plot showing log₂-fold changes in gene expression from bulk RNA-seq comparing human SI^ORG treated with uIFNα2 (x axis) or IFNλ1-like (y axis) to mock-treated controls after 8 h (n = 3). Individual ISGs and selected pro-inflammatory cytokines (red) are highlighted and labeled. f, same as in (c) but for uIFNα2 (left) or IFNλ1 (right) vs. mock-treated human SI^ORG. g, Same as in (e) but for human bronchial^ALI (n = 3). h, Same as in (f) but for GO enrichment analysis of uIFNα2 (left) or IFNλ1 (right) vs. mock-treated human bronchial^ALI.

Extended Data Fig. 8. Pro-inflammatory genes are not broadly induced in bat organoids treated with IFN or infected with RNA viruses.

a, Heatmap showing gene expression log₂-fold changes (from EdgeR bulk RNA-seq differential gene expression analysis) of selected pro-inflammatory genes and selected ISGs in universal IFNα2 (uIFNα2) or IFNλ1 vs. mock-treated human SI^ORG or human Bronchial^ALI (n = 3 each). b, Same as for (a) but for IFN-treated bat SI^ORG (n = 3), bat alv^ORG (n = 3), or bat nasal^ORG (n = 3). c, Same as for (a) but for MARV-, MERS-CoV- or SeV-infected bat alv^ORG (n = 3 each), SeV-infected bat SI^ORG (n = 3), or MARV-infected bat nasal^ALI (n = 3). Comparisons used for calculation of edgeR log₂-fold change are indicated in the legend.

Extended Data Fig. 9. Bat type III IFNλ3-like drive self-regulated antiviral responses in bat SI^ORG.

a, EdgeR differential gene expression analysis of bat SI^ORG (n = 3) treated with IFNλ3-like (LOC107521776) (dose equivalent to 1000 U/ml universal IFNα2) for 8 h compared to mock-treated (n = 3). Each dot represents the average expression (in log₂-count per million, x axis) and log₂-fold change (y axis) of a differentially expressed gene between IFNλ3-like vs. mock-treated SI^ORG. Selected ISGs are highlighted in red and labeled. b, Gene ontology (GO) enrichment analysis of bulk RNA-seq data, performed with clusterProfiler, revealed biological processes significantly enriched among genes upregulated in IFNλ3-like vs. mock-infected bat SI^ORG after 8 h (n = 3 each). The top 10 enriched biological processes are shown. The x-axis indicates the ratio of enriched to total genes within each GO term. Dot size reflects the number of enriched genes per pathway. c, Same as in (a) but for IFNλ3-like (LOC107520938) vs. mock-treated SI^ORG (n = 3). d, Same as in (b) but for IFNλ3-like (LOC107520938) vs. mock-treated SI^ORG. e, RT–qPCR analysis of IFNL3-like (LOC107521776) mRNA, normalized to EEF1A1 (2^–ΔCT) in bat SI^ORG or alv^ORG treated with or without recombinant bat IFNλ3-like (equivalent to 1000 U/ml universal IFNα2) for 8 h. A two-sided Student’s t-test was used to compare IFNL3-like mRNA levels 8 h after treatment to mock-treated controls. ***P<0.001, ****P<0.0001.

Extended Data Fig. 10. 10: Bat type I/III IFN drive antiviral responses with different kinetic profiles in bat SI^ORG or Nasal^ORG.

a, Experimental workflow to assess the temporal induction and maintenance of ISG expression. Bat Nasal^ORG or SI^ORG are treated with universal IFNα2 (uIFNα2) or bat IFNλ1-like for 3 hours, followed by washout and incubation in IFN-free medium for 24 hours. b, Heatmaps showing RT-qPCR analysis of selected ISGs in bat SI^ORG (n = 3), with expression values calculated using the 2 ^-ΔΔCT method: first normalized to EEF1A1 (2^–ΔCT), then to baseline levels at −3 h (before treatment). Timepoints shown include before IFN treatment (−3 h), immediately after treatment (0 h), and various hours post-interferon removal (+6h, +24h). Each heatmap value represents the average normalized ISG expression across three biological replicates. Left, uIFNα2. Right, IFNλ1-like treatment. c, same as in (b) but for bat nasal^ORG. d, RT-qPCR analysis of IFIT1 (normalized to EEF1A1(2^–ΔCT), then expressed as a percentage relative to the average sgScrambled control in bat nasal^ORG engineered with Cas9 and a targeted guide RNA (sgIRF9, sgIFNAR2 or sgIFNLR1) or control guide RNA (sgScrambled) (n = 3 each), treated or not for 8 h with uIFNα2 or bat IFNλ1-like.