Bat organoids reveal antiviral responses at epithelial surfaces

nature immunology Resource https://doi.org/10.1038/s41590-025-02155-1 Bat organoids reveal antiviral responses at epithelial surfaces Received: 1 October 2024 Accepted: 8 April 2025 Published online: 21 May 2025 Max J. Kellner 1,2,3,13 , Vanessa M. Monteil 4,12, Patrick Zelger1,2, Gang Pei5, Jie Jiao 6, Masahiro Onji1,3, Komal Nayak7,8, Matthias Zilbauer 7,8, Anne Balkema-Buschmann9, Anca Dorhoi 5,10, Ali Mirazimi4,11,12 & Josef M. Penninger 1,3,6,13 Check for updates 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. Bats possess a unique ability to host and tolerate pathogens that are highly virulent to humans and nonhuman primates1. Insights from comparative genomic studies in bats have suggested a genetic basis for their exceptional immunity, supported by positive selection or loss of genes that could enhance innate immune responses and limit overt inflammation2–5. However, functional genetic studies in bats remain a challenging task owing to their unique lifestyle, protected status and the limited molecular tools developed and optimized for these non-model organisms6. Pioneering research on bat antiviral immunity has largely focused on peripheral immune responses in infected bats or immortalized cell lines, which have provided crucial insights into their immune defense mechanisms7–9. However, mucosal surfaces, which serve as primary sites for viral entry and form the first line of antiviral defense against both local and systemic infections, have not been thoroughly studied in bats10. In this study, we developed a sustainable organoid platform that accurately models the respiratory and small intestinal (SI) epithelia of Rousettus aegyptiacus (Egyptian fruit bat), a natural reservoir Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria. 2Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria. 3Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria. 4Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden. 5Institute of Immunology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany. 6Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada. 7Wellcome – MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. 8Department of Paediatrics, University of Cambridge, Cambridge, UK. 9Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany. 10Faculty of Mathematics and Natural Sciences, University of Greifswald, Greifswald, Germany. 11National Veterinary Institute, Uppsala, Sweden. 12Public Health Agency of Sweden, Solna, Sweden. 13Helmholtz Centre for Infection Research, Braunschweig, Germany. e-mail: ; 1 Nature Immunology | Volume 26 | June 2025 | 934–946 934 Resource for several human pathogens, including the highly lethal Marburg virus (MARV)7,11–13. Through single-cell RNA sequencing (scRNA-seq), viral infection and genetic perturbation experiments, we uncovered a heightened constitutive expression of innate immune effector genes and enhanced IFN responses to zoonotic viruses in R. aegyptiacus epithelial organoids compared to human counterparts. We further delineated the role of type I and III IFNs in providing robust and long-lasting antiviral protection. These findings establish a valuable resource for studying antiviral immunity at bat epithelial surfaces and reveal species-specific immune adaptations that may underlie bat resilience to emerging zoonotic viruses. Results R. aegyptiacus airway organoids contain diverse cell types The mammalian respiratory epithelium has a central role in orchestrating immune responses to viral infections14. We aimed to generate bat adult stem cell-derived epithelial organoids from the upper and lower respiratory tract of R. aegyptiacus as the model species. To establish a tissue reference dataset, we performed integrative scRNA-seq on whole trachea and lung tissue fragments from a captive-bred R. aegyptiacus and identified distinct clusters of immune, stromal and epithelial cell (EC) lineages (Extended Data Fig. 1a,b and Supplementary Table 1). Among EC clusters, we identified two progenitor stem cell types, namely KRT5+TP63+ basal cells, predominantly found in the trachea, and SFTPC+SFTPB+ alveolar type 2 (AT2) cells, which were exclusively present in the lung (Fig. 1a and Extended Data Fig. 1c). Differentiated epithelial cell lineages included MUC5AC+MUC5B+ secretory goblet and club cells (SCGB1A1, SCGB3A1 and SCGB3A2), ciliated cells (FOXJ1, SNTN and TPPP3), brush cells (POU2F3, AVIL and RGS13) and alveolar type I (AT1) cells (AGER, HOPX and CAV1) (Fig. 1a and Extended Data Fig. 1c). Immunofluorescence staining of bat lung showed that KRT5+ basal cells localized to conducting bronchial and bronchiolar airway structures, while SFTPC⁺ AT2 cells were distributed throughout the lung parenchyma (Fig. 1b). The Egyptian fruit bat respiratory airway epithelium thus contains at least two different progenitor cell types, KRT5+TP63+ basal cells of the upper and lower conducting airway and SFTPC+SFTPB+ AT2 cells of the lung (Fig. 1c). Because a major bottleneck was access to fresh bat tissue, we established a protocol for effective cryopreservation of primary bat tissue, enabling shipping and subsequent use as the starting material for organoid derivation (Methods). Through empirical testing of growth factors known to support the proliferation of adult airway stem cells in vitro15,16, we identified serum-free medium compositions that promoted long-term expansion of basal cell-derived and alveolar cell-derived organoids for at least 6 months (Extended Dat (...truncated)