Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection, with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly, SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.


Introduction
Bats are considered important natural reservoirs for a variety of emerging zoonotic viruses that cause several illnesses in humans and other mammals 1 , including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Hendra virus, Ebola virus, and Marburg virus 2, 3, 4, 5, 6 . The ongoing COVID-19 pandemic is caused by severe acute respiratory coronavirus-2 (SARS-CoV-2) 7 , which also is thought to have originated in bats. This hypothesis is based on multiple studies that demonstrated a high level of genetic similarity between SARS-CoV-2 and several bat-borne coronaviruses such as RatG13 (96.1% identity 7 ) and BANAL-52 (96.8% identity 8 ), which have been detected in bat feces. Studies from a number of different bat species have shown that bat viruses, including coronaviruses, achieve long-term colonization of intestinal tissues 9 . In a study by Watanabe et al. on wild bats captured in the Philippines 10 , enteric coronaviruses were detected in > 50% of the animals, but clinical signs of disease were absent. Similarly, Subudhi et al. found that 30% of North American little brown bats harbored coronaviruses in their intestines but did not display any signs of illness 11 . Tong et al. analyzed rectal swabs and intestinal tissues from asymptomatic fruit bats in Peru and identi ed a novel in uenza A virus, H18N11 12 . In contrast to bats, where gastrointestinal infections with eukaryotic viruses are frequent and are commonly asymptomatic 13 , a similar colonization of the human gut with non-pathogenic eukaryotic viruses has not been reported, pointing to species-speci c mechanisms 14 .
Studying coronavirus infection in the GI tracts of bats is di cult, since few institutions maintain bat colonies for in vivo infection experiments, and cell lines from the GI tract of bats are not available, limiting in vitro analyses 15,16 . Organoid cultures have untapped potential as a model to study the mechanisms of viral infection in bat cells in vitro 17,18 . Organoids are permanent three-dimensional cultures that replicate the physiological and functional characteristics of their tissues of origin and that allow controlled studies of complex primary GI epithelial tissues in vitro 19 . Organoids from various human and murine tissues have been developed from tissue-derived stem cells and have been successfully used to investigate a wide range of disease processes, including viral infections 17,18,20,21 . Importantly, growth conditions for GI organoids appear similar across multiple species 22 . Two previous studies have described the generation of intestinal organoid cultures from bat species 23,24 . Intestinal organoids developed from Chinese horseshoe bats, Rhinolophus sinicus, showed susceptibility to SARS-CoV-2, but lacked long-term active proliferation past 4-5 weeks 25 . Intestinal organoids derived from Leschenault's rousette, Rousettus leschenaultii. showed susceptibility to Pteropine orthoreovirus, but not SARS-CoV-2 23 . However, neither of these studies evaluated the cellular antiviral mechanisms of bat organoid tissues 23,25 .
The hypothesis that altered IFN responses in bats compared to other species promote increased viral tolerance is a central paradigm in bat immunology 26, 27 . In Australian black ying foxes (P. alecto), a high level of constitutive IFN-α expression was detected, which has led to the concept that an "always on" IFN signaling system in bats can effectively suppress viral replication and prevent disease early on after infection 26,28 . Increased basal gene expression in bats also was described for several other genes involved in innate viral recognition and response, including IRF1, IRF3 and IRF7 29 and the ISG oligoadenylate synthase 1 (OAS1) 30 . Conversely, dampened activation of stimulator of IFN genes (STING), a nucleic acid sensor involved in the regulation of IFN expression upon viral infection, also has been reported 31,32 . Importantly, these characteristics of the IFN system appear to be unique to particular bat species, pointing to a need for more detailed analyses.
Jamaican fruit bats (JFBs) are thought to be natural carriers of zoonotic viruses such as rabies, West Nile and dengue viruses and are one of the most common bats in the Americas, making them a relevant species for experimental investigations 6, 33,34,35,36,37 . JFBs also are susceptible to experimental infection with Zika virus and MERS-CoV 6, 36 . Based on the recently annotated genome 38, 39 , JFBs have one interferon (IFN)-β gene, ve IFN-α genes, and ve IFN-ω genes. Multiple interferon regulatory factors (IRFs) have also been identi ed, making JFBs a tractable model system for studies of antiviral immunity.
Here we established and characterized gut organoids from JFBs to study the susceptibility and immune response of the JFB intestinal epithelium to SARS-CoV-2 infection. We found that JFB intestinal epithelial cells supported modest viral replication that did not result in the release of infectious virions or cytopathic effects. Contrary to the "always on" paradigm for antiviral interferon responses in bats 28 , the organoids mounted a robust interferon response to infection with active SARS-CoV-2. Proteomics and pathway analysis revealed that the JFB organoid proteome pro les matched pro les found in other SARS-CoV-2 infection studies and that SARS-CoV-2 infection activated innate in ammatory and cellular repair responses in this model system.

Results
Development and Characterization of JFB Gastrointestinal Organoids.
We established JFB gastrointestinal organoid cultures from fresh and cryopreserved stomach and from proximal and distal small intestine ( Fig. 1A and Supplemental Fig. 1A, B). Organoids formed within one day of crypt/gland isolation and were successfully maintained in a simple growth medium containing DMEM and 50% L-WRN-conditioned medium (Supplemental Fig. 1C).The murine noggin, R-spondin, and Wnt3a secreted by the L-WRN cells 40 show protein sequence similarities of 98%, 86%, and 99% with the orthologous JFB proteins, suggesting that these factors would be active in JFB cells (Supplemental Fig. 2).
Established JFB organoids mimicked the epithelial structure of JFB gastrointestinal tissue, with a simple columnar epithelium, a basal nucleus and a de ned luminal space (Fig. 1B, Supplemental Fig. 3A, B). Mucus-secreting cells were present in organoids derived from distal small intestine and stomach, but were rare in proximal small intestinal organoids, consistent with the cellular composition of the respective tissues of origins (Fig. 1B, Supplemental Fig. 3A, B). Morphometric analysis with OrganoSeg 41 showed that organoid size varied between different passages, but with no clear trends, and organoid shape also did not change signi cantly over six consecutive passages (Fig. 1C). JFB organoids were maintained for at least 30 passages (> 6 months), and also were successfully cryopreserved and re-cultured from cryopreserved stocks (data not shown).
We next performed transcriptional analysis of the organoids to con rm tissue-speci c gene expression patterns. The distal and proximal intestinal organoids expressed the intestine-speci c genes Vil1, Cdx2, and Muc2, while the gastric organoids showed increased expression of the chief cell marker pepsinogen C (Pgc) with low expression of Vil1, Cdx2 and Muc2 ( Fig. 2A). Since no speci c reagents for JFB cells are available, we next performed an unbiased proteome analysis using data-independent acquisition (DIA) mass spectrometry using organoids from JFB distal small intestine. Several key proteins characteristic of small intestinal epithelial cells in other mammals such as villin, E-cadherin, keratin 18 and 19, Na + /K + ATPase, claudin 18, and a mucin (MUC5AC-like) were detected ( Fig. 2B) 42 , con rming the identity of the intestinal organoids. Measurement of transepithelial electrical resistance (TEER) across organoid monolayers seeded on transwell inserts showed that the gastrointestinal organoids established a physiological epithelial barrier, with the stomach having the highest TEER compared to the intestinal organoids (Fig. 2C). Confocal imaging analysis of cytokeratin expression con rmed epithelial cell polarization and correct inside-in orientation of the organoids (Fig. 2D). Collectively, these analyses demonstrate that gastrointestinal organoids from JFBs replicate key features of the gastrointestinal epithelium.
Infection of JFB distal organoids with SARS-CoV-2 leads to replication of viral genomes.
To determine whether the JFB intestine supports SARS-CoV-2 infection, organoids were dissociated and then inoculated with SARS-CoV-2 at MOIs of 0.1, 1 and 10. We selected distal intestinal organoids for these experiments, based on several previous publications that demonstrated SARS-CoV-2 replication in human ileal organoids 43,44,45 . Quantitative PCR analysis of viral genomes in JFB organoid cell lysates revealed a signi cant, concentration-dependent increase (> 1 log, P ≤ 0.05) in SARS-CoV-2 gene E RNA at similar increase at an MOI of 1 at 48 hpi (Fig. 3B). Importantly, signi cant expression of subgenomic (sg)RNA (> 2 log-fold above baseline) for gene E indicating active viral replication in the organoids also was identi ed 46 , albeit at low levels ( Fig. 3C). However, plaque assays performed on the culture supernatants failed to detect the presence of infectious SARS-CoV-2 above baseline values derived from the inoculum, suggesting incomplete or ineffective viral replication or failure to secrete progeny virus (Fig. 3D). Culture of the organoids in differentiation medium with reduced Wnt3a or as 2D monolayers did not alter these results (data not shown). Notably, SARS-CoV-2 incubation in medium for 48 h did not impact detection of viral copy numbers by PCR, but did reduce the viral titer measured by plaque assay by > 1 log-fold, suggesting a loss of infectivity over time (Supplemental Fig. 4). Interestingly, immuno uorescence analysis of SARS-CoV-2 spike protein in infected JFB organoids revealed only a few positive cells, and these cells were not associated with morphologically intact organoids (Fig. 4E).

Lack of cytopathic effect but increased growth in SARS-CoV-2 infected JFB organoids
We also evaluated the cell viability of JFB distal intestinal organoids following SARS-CoV-2 infection by measuring caspase-3 activity with NucView® 47 (Fig. 4A). In Vero E6 cells, infection with SARS-CoV-2 induced a strong upregulation of caspase-3, consistent with the well-characterized cytopathic effect of the virus in this cell type. A small number of apoptotic cells were present in all JFB organoid cultures, likely due to physiological cell turnover. However, in contrast to observations in Rhinolophus sinicus organoids 25 , SARS-CoV-2 did not appear to have a cytopathic effect in JFB organoids (Fig. 4A,B To determine whether active viral infection was responsible for the observed induction of antiviral and in ammatory genes, or whether gene expression was induced by unspeci c activation of pattern recognition receptors, we also treated the JFB organoids with a panel of TLR agonists targeting TLR2, 3, 7, and 9 and with UV-inactivated SARS-CoV-2 for 48 h. Notably, stimulation with TLR2/1 and TLR3 agonists led to increased expression of interferon and in ammatory cytokines 6 h post inoculation (Supplemental Fig. 5). However, no signi cant upregulation of these genes was observed with any of the stimuli at 48 h ( Fig. 5E-H). These observations suggest that active infection with SARS-CoV-2 is required for sustained upregulation of antiviral and in ammatory gene expression.

Impact of SARS-CoV-2 infection on the JFB intestinal epithelial cell proteome
A quantitative proteomic work ow based on data-independent acquisition (DIA) mass spectrometry was used to perform a comprehensive analysis of the cellular responses of JFB organoids to SARS-CoV-2 infection. The DIA analysis of SARS-CoV-2-infected and mock infected enteroids after 48 h yielded a total of 8,321 proteins and protein isoforms, based on protein FASTA les retrieved from the A. jamaicensis reference genome 51,52 . Interestingly, all detected proteins were present in both experimental conditions. A comparative analysis of mock-infected and SARS-CoV-2 infected JFB organoids revealed 63 upregulated and 155 downregulated proteins, including isoforms, with a ≥ 2-fold change at P ≤ 0.05 ( Fig. 6A and Supplemental Table 1). To better understand antiviral responses in the JFB intestine, we next compared the identi ed proteins to a comprehensive list of human interferon-stimulated genes (ISGs, Supplemental Table 2) 53 . Interestingly, 100 of all identi ed JFB proteins could tentatively be classi ed as ISGs.
However, only one of the ISG proteins, ribonucleases P/MRP protein subunit POP1 (POP1), was signi cantly upregulated in response to SARS-CoV-2, while four ISG proteins (ERLEC1, CFB, ARMCX3 and ITIH2) were signi cantly downregulated (Fig. 6B). Overall, top upregulated proteins, based on fold change in abundance, were hepatocyte growth factor-like protein/macrophage stimulatory protein (HGFL/MST1), CUB domain-containing protein 1-like, acyl-CoA-binding domain-containing protein 5 (ACBD5), ketosamine-3-kinase (KT3K) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1, Fig. 6C). Top down-regulated proteins included BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 3 (KCTD10), CSC1-like protein 1 (TMEM63A), nuclear complex protein 3 homologue, histone H2A-β, and cell division complex protein 45 homologue (CDC45) (Fig. 4D). Several of these proteins are involved in regulation of cell turnover and posttranslational modi cations. We next performed Ingenuity Pathway Analysis (IPA) and Enrichr analysis 54 to assess more complex functional changes induced by SARS-CoV-2. IPA revealed acute phase response signaling, a key innate pathway triggered by infection and injury, as the most signi cantly regulated pathway, followed by Apelin liver signaling 55 , which is involved in intestinal in ammation, repair, and wound healing (Fig. 6D). Top regulated cellular functions were cell assembly, organization, maintenance, movement, signaling and morphology (Fig. 6D). These ndings suggest that SARS-CoV-2 triggers regenerative response pathways, consistent with the increased organoid size observed in the SARS-CoV-2-infected compared to mockinfected cultures. Similarly, Enrichr identi ed signi cant upregulation of pathways associated with cell viability and differentiation, such as PI3/AKT signaling and the longevity regulating pathway, along with signatures associated with intestinal epithelial infection and chemokine signaling when using the human 2021 KEGG pathways database (Fig. 6E). Importantly, Enrichr analysis also found multiple signi cant matches for protein signatures that were previously found to be upregulated in SARS-CoV-2 infection in various experimental systems 56, 57, 58, 59, 60 (Fig. 6F). Overall, the proteomics analysis points to the activation of innate in ammatory and regenerative pathways along with characteristic COVID-19 signatures upon SARS-CoV-2 infection of the JFB intestinal epithelium.

Discussion
In this study, we established and characterized organoid cultures from the proximal and distal small intestine and stomach of JFBs.  35,69 . While we did not detect an upregulation in anti-apoptotic factors in our proteome screen of SARS-CoV-2-infected JFB organoids, we found a signi cant upregulation of pathways associated with cell growth and repair, including apelin liver signaling and wound healing signaling. These observations were consistent with the increase in organoid size and organoid formation that we detected by microscopic analysis and indicate activation of growth and repair pathways in response to SARS-CoV-2 infection. Taken together, our ndings suggest that bat organoids activate protective repair pathways upon viral infection that may enable the bats to tolerate viral infection in the absence of tissue damage and associated clinical signs.
We used a proteomics approach to gain deeper insights into the cellular responses induced in the SARS-CoV-2 infected JFB intestinal epithelium. Notably, our study was the rst, to our knowledge, to use a DIAbased proteomics approach with JFB cells. Our analysis con rmed the identity of the organoids as small intestinal epithelial cells based on expression of key enterocyte markers. Consistent with the increased gene expression of pro-in ammatory cytokines in SARS-CoV-2-infected JFB organoids that we detected, in ammatory pathways including the acute phase response and chemokine signaling also were induced at the protein level. Conversely, although SARS-CoV-2 infection induced expression of type I interferon transcripts in JFB organoids, no signi cant increase in ISGs was detected on the protein level. This lack of ISG regulation is inconsistent with proteomics results obtained in SARS-CoV-2-infected human Calu-3 cells, which showed a strong induction of the antiviral ISG signature 70 , and may re ect a JFB-speci c disconnect between transcriptional activation of interferons and downstream ISGs that warrants further studies. Alternatively, downregulation of ISG proteins may have been caused by active downregulation of antiviral ISG pathways by SARS-CoV-2 accessory proteins. Importantly, Enrichr analysis also revealed that some of the activated pathways matched those identi ed by other studies on SARS-CoV-2 infection. Notably, there are several limitations to the proteomics approach undertaken in our study. First, the nontargeted DIA approach may not be sensitive enough to identify strongly regulated targets with a low overall expression level 71 . Second, an annotated proteome of the JFB is currently not available and thus had to be inferred from the genome, which may lead to misidenti ed proteins. Lastly, pathway analysis was based on human databases, which again may miss JFB-speci c signaling pathways.
Importantly, we successfully validated JFB organoids as an experimental tool and demonstrated that these JFB organoids can be maintained long term without the need for bat speci c growth factors. Wnt, noggin and R-spondin are highly conserved in mammalian species, with a high degree of sequence identity between mice and JFBs. The growth requirements for our JFB organoids are consistent with growth conditions previously described for Chinese horseshoe bats 25 and Rousettus bats 23 . Similar culture conditions also have been successfully used to culture intestinal organoids from cat, dog, cow, horse, pig and sheep 22 . We demonstrate that JFB organoids from stomach, proximal and distal small intestine recapitulate the histology and morphology of the tissue of origin, with polarized columnar epithelial cells, mucus secretion, development of an intact epithelial barrier and expression of tissuespeci c genes. Thus, we have developed and validated a new research tool that will allow experimental analysis of the physiology and function of the gastrointestinal epithelium of Jamaican fruit bats in future studies.
To summarize, we established and characterized JFB gastrointestinal organoids that recapitulated the organ-speci c multicellular composition of JFB gastrointestinal tissue. We demonstrated SARS-CoV-2 sgRNA replication at a low e ciency in JFB distal intestinal organoids via qPCR but were unable to detect release of infectious virus. SARS-CoV-2 infection induced a robust upregulation of interferons and proin ammatory genes in the organoid cells. Moreover, SARS-CoV-2 infection of JFB organoids led to increased growth and activation of cellular regeneration and healing pathways, which might contribute to the improved viral tolerance in this bat species.

Materials And Methods
Tissue samples. Male and female Jamaican fruit bats (Artibeus jamaicensis) were maintained as a breeding colony in an AAALAC-accredited facility at Colorado State University (CSU) under and approved Institutional Animal Care and Use Committee protocol (#1034). For organoid derivation, ve adult bats (4 male, 1 female) were euthanized by 5% iso urane in O 2 followed by thoracotomy. The gastrointestinal tracts were harvested in RPMI-1640 medium and were shipped overnight on ice from CSU to Montana State University (MSU).
Crypt and gland isolation methods. Bat tissues were processed immediately upon arrival or were cryopreserved and then thawed rapidly if needed 72 . To derive organoids, proximal intestinal and distal intestinal tissues were washed in cold PBS and cut into ~ 1mm pieces. The minced tissue was incubated in 15 mM EDTA in PBS supplemented with, penicillin, streptomycin, and Fungizone (GE Healthcare Life Sciences) with gentle shaking for 10 min increments until crypts appeared in the supernatant. Large tissues pieces were removed by sedimentation. The supernatant containing the crypts was transferred into a new 50 mL tube and pelleted by centrifugation for 8 min at 150 g. Gastric tissues were digested using a digestion solution containing 5 U/mL collagenase type IV and 0.2 mg/mL DNAse (both Sigma-Aldrich), following our published protocols 73,74 . Recovered crypts/glands were resuspended in 10 µl of Matrigel and plated in 96-well plates. After the gel was polymerized, 200 µl of medium (Supplemental Table 3) was added, and the plates were incubated at 37°C with 5% CO 2 for one week.
Maintenance of JFB organoids. For passaging, the Matrigel patties containing organoids were digested for 3 min in TrypLE (Gibco) at 37˚C and pipetted up and down 50 times. The digested organoids were harvested by centrifugation for 5 min, 200 g at 4˚C, then were resuspended Matrigel and plated in a 24well plate. After the gel had polymerized, 500 µl of medium was added and the plate was incubated at 37˚C with 5% CO 2 . The medium was changed every other day and the organoids were passaged every 5-7 days.
Optimization of growth conditions. In addition to the basic growth medium, termed L-WRN medium, described above, we also tested a commercially available growth medium, IntestiCult™ (StemCell), a complex medium termed "colonoid medium" described by Tsai et al. 72 , and analyzed medium supplementation with a number of different growth factors commonly used in organoid culture protocols (Supplemental Table 3). We prepared a medium with all available growth factors (L-WRN Plus) and then eliminated one reagent at a time from L-WRN Plus to determine the in uence of the reagent on organoid growth. For this assay, the organoids were digested with TrypLE for 3 min and plated in a 96-well plate with the different media. Cell viability and proliferation were measured using the CellTiter-Glo luminescence assay (Promega).
Histological Analysis of JFB Organoid Cultures. Organoids were recovered from the culture plates and treated with Histogel (ThermoFisher) prior to formalin xation and para n embedding, following standard protocols. Slides were stained with hematoxylin/eosin and with Alcian Blue to visualize mucus production.
SARS-CoV-2 Infection of JFB organoids. Bat organoids were dissociated by incubation with 350 µL TrypLE to expose the apical and basolateral epithelial surface to the virus. Dissociated organoids were transferred to a BSL3 laboratory and then inoculated with SARS-CoV-2 (strain USA-WA1/2020, BEI Resources), at a multiplicity of infection (MOI) of 0.1, 1 and 10 for 2 h at 37°C with frequent gentle agitation. Notably, the SARS-CoV-2 strain used was shown to have a defective furin cleavage site 75 , but readily infected inducible pluripotent stem cell-derived human intestinal organoids in control experiments. The infected bat organoids were incubated in 30 µL of DMEM at 37°C for 2 hours with occasional shaking. Organoids were collected into 500 µL DMEM and centrifuged at 200 g for 5 min to wash. Then cells were resuspended in 30 µL Matrigel and plated. After 10 min to allow gelation of the Matrigel, medium was added to the organoids. This medium was removed and fresh medium added to eliminate free viral particles. Then the plates were incubated at 37°C for the indicated intervals. Infectious particles in culture supernatants were detected for each time point by plaque assay on Vero E6 cells, as previously described 76 .
Quantitative RT-PCR. To analyze gene expression and cell-associated viral RNA, RNA was extracted from organoids using the Direct-zol RNA Miniprep-Plus (Zymo Research). The RNA was converted to cDNA using iScript Reverse Transcription Super mix for RT-qPCR (BioRad). Primers for gastric and intestinal epithelial cell-speci c genes and cytokines were designed using NCBI primer blast using the JFB genome (Artibeus jamaicensis, textid: 9417) and are listed in Supplemental Table 4. GAPDH was ampli ed as housekeeping gene in each PCR reaction. For each gene, a standard curve was created, and gene copy numbers for each gene of interest were normalized to the copy numbers of the housekeeping gene, GAPDH. To quantify SARS-CoV-2 in the organoid supernatant, viral RNA was extracted from culture supernatants using the QIA®Amp Viral RNA Mini kit (Qiagen). Viral genomes were then quanti ed in a single step RT-PCR reaction using primers and a TaqMan probe to the SARS-CoV-2 envelope (E) gene, as previously described 76 , and the Quanta Bio ToughMix Master Mix. In addition, a forward primer to the leader sequence was used together with the reverse primer and probe to detect E gene sgRNA as described by Wölfel et al. 46 . An RNA standard curve generated from a T7 in vitro transcribed gBlock™ sequence (Integrated DNA Technologies) was used for normalization.
Immuno uorescence Staining. For visualization of epithelial cytokeratin, we used a mouse-anti cytokeratin antibody that detects cytokeratins in a wide range of species (Thermo sher, 50-191-151). For visualization of SARS-CoV-2 protein in the organoid cultures, a monoclonal antibody to SARS-CoV-2 (11G10-F8) was generated in house, using a standard hybridoma protocol 77 . Brie y, mice were immunized with 10 µg UV-inactivated SARS-CoV-2 (USA-WA1/2020) 76 in Titermax adjuvant (Sigma) three times separated by at least two weeks. 11G10-F8 was then generated from a fusion of mouse splenocytes with SP2/0 cells. Mouse sera were screened for reactivity to the virus by ELISA. Clone 11G10-F8 recognizes the RBD region of the S1 subunit of the spike protein and was used at a concentration of 10 µg/mL. For immuno uorescence analysis, organoids were xed with 4% PFA, permeabilized with 0.2% Triton X-100, and then treated with blocking buffer (DPBS with 10% FBS, 0.2% Triton X-100, 0.1% BSA, and 0.05% Tween) overnight. After washing, samples were incubated with primary antibody for 2 hours at room temperature. Then the secondary antibodies (goat anti-mouse IgG (H + L) AlexaFluor 594, Invitrogen, A11005; or rat anti-mouse IgG1 eFluor660, eBiosciences, 50-112-4348), were added at 1:100 and incubated for 2 hours at room temperature. The nuclei were stained with 5 µM DAPI (MP Biomedicals, 0215757405). Actin laments were stained with ActinGreen 488 ReadyProbes reagent-(Invitrogen, R37110). Stained organoids were imaged on an inverted SP5 Confocal Scanning Laser Microscopy (Leica) with 405 nm, 488 nm, 561 nm and 633 nm laser excitation lines using a 20x objective (W 2010; Zeiss, Oberkochen, Germany). Z-stacks of 2-11 randomly selected organoids with intact morphology for each experiment and condition were recorded.
Cell viability and organoid growth. To measure caspase 3 activity in SARS-CoV-2-infected organoids, NucView488 (Biotium) was added to the medium at 3 µM once the organoids were re-plated following incubation with the virus. For measuring caspase-3 activity, the organoids were imaged using Life Technologies EVOS FL Auto system with a 10x objective. The images were analyzed using ImageJ version 1.48V and NucView positive pixels were counted automatically on the thresholded images.
Bright eld images of the organoid cultures were used to measure organoid size for normalization of the NucView data and for assessment of organoid growth.

Proteomics analyses
Triplicate samples of distal intestinal organoids were infected with SARS-CoV-2, MOI 10, for 48 h as described above and then were lysed in RIPA lysis buffer (25 mM Tris/Cl, 150 mM NaCl, 1% NP-40, 1% SDS, 1% protease inhibitor) by passing the samples through a 26.5G needle 5 times on ice. Samples were stored at -80° C until they were analyzed at the IDeA National Resource for Quantitative Proteomics. An Orbitrap Exploris 480 was used for data-independent acquisition (DIA) mass spectrometry with a 60 min gradient per sample and gas-phase fractionation to obtain comprehensive proteomic pro les of the organoids. Chromatogram libraries were constructed using Prosit 78 , and proteins were identi ed and quanti ed using EncyclopeDIA, based on protein FASTA les retrieved from NCBI RefSeq for the Jamaican fruit bat (BioProject PRJNA673233) 51,52 . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 79 partner repository with the dataset identi er PXD036016. False discovery thresholds of 1% were applied. The ProteiNorm app was used to optimize data normalization 80 , and Scaffold DIA (Proteome Software, Portland, OR) was used for visualization. The MS2 exclusive intensities were normalized using cyclic loess and linear models for microarray (limma) and lm t with empirical Bayes smoothing was used for the analysis 81 . Proteins with an FDR-adjusted P-value ≤ 0.05 and an absolute fold change ≥ 2 were considered signi cant. Ingenuity Pathway Analysis (Qiagen) and Enrichr 54 with combined score ranking (c = log(p) * z, where c = the combined score, P = Fisher exact test P-value, and z = z-score) were used to identify cellular signaling pathways. COVID-19 related gene sets identi ed by Enrichr were based on the "The COVID-19 Drug and Gene Set Library, 2021 version" website 82 . To analyze the impact of SARS-CoV-2 infection on ISGs, proteins identi ed in the JFB organoids were compared to a comprehensive list of ISGs 53