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

Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection Marziah Hashimi, Thomas Sebrell, Jodi Hedges, Deann Snyder, Katrina Lyon, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2340919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Immunology/Infectious diseases/Viral infection Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 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 identified a novel influenza 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-specific mechanisms 14 . Studying coronavirus infection in the GI tracts of bats is difficult, 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 flying 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, five IFN-α genes, and five IFN-ω genes. Multiple interferon regulatory factors (IRFs) have also been identified, 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 profiles matched profiles found in other SARS-CoV-2 infection studies and that SARS-CoV-2 infection activated innate inflammatory 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. 1 A 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 defined luminal space (Fig. 1 B, 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. 1 B, 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 significantly over six consecutive passages (Fig. 1 C). 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 confirm tissue-specific gene expression patterns. The distal and proximal intestinal organoids expressed the intestine-specific 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. 2 A). Since no specific 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. 2 B) 42 , confirming 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. 2 C). Confocal imaging analysis of cytokeratin expression confirmed epithelial cell polarization and correct inside-in orientation of the organoids (Fig. 2 D). 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 significant, concentration-dependent increase (> 1 log, P ≤ 0.05) in SARS-CoV-2 gene E RNA at 48 and 72 hours post infection (hpi, Fig. 3 A). The SARS-CoV-2 PCR in culture supernatants showed a similar increase at an MOI of 1 at 48 hpi (Fig. 3 B). Importantly, significant expression of subgenomic (sg)RNA (> 2 log-fold above baseline) for gene E indicating active viral replication in the organoids also was identified 46 , albeit at low levels (Fig. 3 C). 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. 3 D). 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, immunofluorescence 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. 4 E ) . 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. 4 A). 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. 4 A,B), since the proportion of apoptotic cells did not change upon infection. Interestingly, SARS-CoV-2 caused a significant increase in organoid size and in the number of organoids that had re-formed from single cells after 48 h of infection (Fig. 4 C,D), indicating that viral infection triggered increased cell proliferation in the bat intestinal epithelium. SARS-CoV-2 induces expression of type I interferons and proinflammatory cytokines in JFB organoids. Unique characteristics of the interferon (IFN) system have been linked to the increased viral tolerance observed in many bat species 26 . Therefore, we used quantitative RT-PCR to analyze gene expression of type I interferons and proinflammatory cytokines in JFB distal small intestinal organoids following 48 h exposure to SARS-CoV-2. As shown in Fig. 5 A, expression of type I interferon Ifna4l was upregulated at 48 hpi with an MOI of 10, while an MOI of 1 caused significant upregulation of the gene at 72 hpi. Gene expression of Ifnb also was significantly increased with both MOIs at 48 hpi and remained elevated with the lower viral dose at 72 hpi (Fig. 5 B). Type III IFNs are known to play a role in mucosal antiviral immunity and SARS-CoV-2 infection and also may have unique functions in bats 48 , 49 , 50 . However, the type III IFN loci in JFBs are poorly annotated 38 , 39 , and we unable to generate functional primers based on the published genome. Interestingly, organoid infection with SARS-CoV-2 at an MOI of 1 significantly increased expression of the proinflammatory cytokines Tnf and Il6 at 48 hpi, and Il6 remained elevated at 72 hpi (Fig. 5 C,D). The above data suggest that JFB distal organoids exhibited an anti-viral and pro-inflammatory response to SARS-CoV-2 infection. To determine whether active viral infection was responsible for the observed induction of antiviral and inflammatory genes, or whether gene expression was induced by unspecific 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 inflammatory cytokines 6 h post inoculation ( Supplemental Fig. 5 ). However, no significant upregulation of these genes was observed with any of the stimuli at 48 h (Fig. 5 E-H). These observations suggest that active infection with SARS-CoV-2 is required for sustained upregulation of antiviral and inflammatory gene expression. Impact of SARS-CoV-2 infection on the JFB intestinal epithelial cell proteome A quantitative proteomic workflow 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 files 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. 6 A and Supplemental Table 1 ). To better understand antiviral responses in the JFB intestine, we next compared the identified proteins to a comprehensive list of human interferon-stimulated genes (ISGs, Supplemental Table 2 ) 53 . Interestingly, 100 of all identified JFB proteins could tentatively be classified as ISGs. However, only one of the ISG proteins, ribonucleases P/MRP protein subunit POP1 (POP1), was significantly upregulated in response to SARS-CoV-2, while four ISG proteins (ERLEC1, CFB, ARMCX3 and ITIH2) were significantly downregulated (Fig. 6 B). 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. 6 C). 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. 4 D). Several of these proteins are involved in regulation of cell turnover and posttranslational modifications. 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 significantly regulated pathway, followed by Apelin liver signaling 55 , which is involved in intestinal inflammation, repair, and wound healing (Fig. 6 D). Top regulated cellular functions were cell assembly, organization, maintenance, movement, signaling and morphology (Fig. 6 D). These findings suggest that SARS-CoV-2 triggers regenerative response pathways, consistent with the increased organoid size observed in the SARS-CoV-2-infected compared to mock-infected cultures. Similarly, Enrichr identified significant 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. 6 E). Importantly, Enrichr analysis also found multiple significant 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. 6 F). Overall, the proteomics analysis points to the activation of innate inflammatory 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. Using this model, we investigated the response of JFB intestinal epithelial cells to infection with SARS-CoV-2. Considering that JFBs are susceptible to MERS-CoV, Zika virus, and rabies virus 6 , 36 , 37 , we evaluated the susceptibility of the JFB distal intestinal organoids to SARS-CoV-2. Notably, JFBs are not thought to be natural carriers of SARS-CoV-2, and no studies on in vivo infection of JFBs with SARS-CoV-2 have been published to date. Considering the vast number and associated genetic diversity of bat species it is not surprising that SARS-CoV-2 infection experiments in other bat species have yielded conflicting results. In Egyptian fruit bats ( Rousettus aegyptiacus) , transient asymptomatic respiratory tract infection with viral replication in lung and trachea and oral and fecal shedding was achieved upon experimental SARS-CoV-2 inoculation 61 . Conversely, American big brown bats ( Eptesicus fuscus ) appeared resistant to infection with SARS-CoV-2 62 . Likewise, intestinal organoids derived from two different bat species responded differently to SARS-CoV-2 infection. Organoids from Chinese horseshoe bats, where SARS-CoV-2-like virus has been detected 7 , produced infectious SARS-CoV-2 virions at similar levels as human intestinal organoids 25 . In contrast, intestinal organoids from Leschenault's rousette bats ( Rousettus leschenaultii ) failed to support SARS-CoV-2 replication 23 . Interestingly, PCR analysis revealed a significant increase in viral and sgRNA in the JFB distal organoids at 48 and 72 hpi, which demonstrates initiation of viral replication in the organoids. SARS-CoV-2 genomes also were significantly increased in organoid culture supernatants. Using immunohistochemistry, we detected SARS-CoV-2 spike protein in individual cells, but not in morphologically intact JFB organoids. This observation may reflect shedding of viable virus-infected cells from the epithelial monolayer, as described for other viral infections 63 . We also did not detect infectious virions in organoid cells or supernatants using plaque assays in VeroE6 cells, suggesting that JFB intestinal organoids support incomplete SARS-CoV-2 infection. A similar limited and incomplete replication of SARS-CoV-2 was also reported in cell lines from several different bat species, even after transduction with human ACE2, in a recent study by Aicher et al. 64 . However, the presence of sgRNA and of SARS-CoV-2 protein in some cells suggest that entry and replication of the virus did occur in the JFB organoids. This interpretation is consistent with a study by Yan et al. that predicted a moderate ability of SARS-CoV-2 to infect JFB cells based on the protein sequence of the SARS-CoV-2 receptor ACE2 65 and our unpublished observations of ACE-2 gene expression on the JFB organoids. Loss of the furin cleavage site in the WA01 reference stain of SARS-CoV-2 also may have had an impact on the efficacy of infection 66 . Further experiments are needed to evaluate at which stage of viral replication cycle SARS-CoV-2 replication stalls in the JFB organoid model and whether JFBs are permissive to SARS-CoV-2 infection in vivo . Notably, many previous studies on viral infection in bats have relied solely on viral nucleic acids to measure infection 5 , 10 , 11 , 12 , 35 , 62 . Therefore, it is difficult to assess whether the failure to detect replication-competent virions was unique to our infection model. Our results demonstrate that active SARS-CoV-2 virus induced a robust anti-viral immune response, with increased expression of IFN-α and IFN-β at 48 h after SARS-CoV-2 infection. This strong induction of interferons in response to viral infection was surprising, since the current paradigm is that the interferon system in bats is constitutively active, based on studies in Australian black flying foxes ( P. alecto) 26 , 67 . Conversely, potent interferon responses were detected in serotine bats ( Eptesicus serotinus ) and David’s myotis bat cells upon SARS-CoV-2 infection 64 . These observed differences point to species-specific immune system characteristics in bats, consistent with the high level of genetic diversity in the order Chiroptera, which comprises over 1,400 species. The lack of a cytopathic effect in SARS-CoV-2-infected JFB organoids was an intriguing observation. It has been shown that SARS-CoV-2 causes neither apoptotic nor necrotic cell death in the gastrointestinal tract of infected human patients. Whether SARS-CoV-2 impacts viability of organoid cultures is still a matter of debate. Lamers et al. 43 observed increased apoptotic cell death in human enteroids at 60 hpi, and Zhou et al. 25 state that both human and horseshoe bat enteroids developed a cytopathic effect after SARS-CoV-2 inoculation. Conversely, studies by Stanifer et al. 48 and Zang et al. 68 did not describe increased cell death in human SARS-CoV-2 infected enteroids. Data from several studies in bat cells suggest that heightened IFN responses in these cultures may prolong viral infection by limiting pathogen-induced cell death through induction of anti-apoptotic genes including BCL-2 and PMAIP1 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 significant 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 findings 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 first, to our knowledge, to use a DIA-based proteomics approach with JFB cells. Our analysis confirmed 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-inflammatory cytokines in SARS-CoV-2-infected JFB organoids that we detected, inflammatory 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 significant 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 reflect a JFB-specific 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 identified by other studies on SARS-CoV-2 infection. Notably, there are several limitations to the proteomics approach undertaken in our study. First, the non-targeted 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 misidentified proteins. Lastly, pathway analysis was based on human databases, which again may miss JFB-specific 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 specific 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 tissue-specific 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-specific multicellular composition of JFB gastrointestinal tissue. We demonstrated SARS-CoV-2 sgRNA replication at a low efficiency 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 pro-inflammatory 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, five adult bats (4 male, 1 female) were euthanized by 5% isoflurane 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 24-well 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 influence 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 fixation and paraffin 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 . Treatment of JFB organoids with TLR agonists and inactivated virus. To analyze transcriptional response of JFB organoids to stimulation with pathogen-associated molecular patterns, organoids were trypsinized and then re-embedded into Matrigel in the presence of the following TLR agonists (Human TLR1-9 agonist kit, InvivoGen): TLR1, Pam3CSK4, 1 µg/mL; TLR2, heat-killed Listeria monocytogenes (10 8 /mL); TLR3, low molecular weight poly I:C, 10 µg/mL; TLR7, imiquimod, 1 µg/mL; TLR9, ODN2006, 5 µM. Alternatively, organoids were treated with UV-inactivated SARS-CoV-2 76 (10 µg/mL). After 48 h, Organoids were lysed in TRI Reagent (Sigma) and processed for RNA isolation and RT-PCR. 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-specific 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 amplified 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 quantified 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. Immunofluorescence Staining. For visualization of epithelial cytokeratin, we used a mouse-anti cytokeratin antibody that detects cytokeratins in a wide range of species (Thermofisher, 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 . Briefly, 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 immunofluorescence analysis, organoids were fixed 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 filaments 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. Brightfield 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 profiles of the organoids. Chromatogram libraries were constructed using Prosit 78 , and proteins were identified and quantified using EncyclopeDIA, based on protein FASTA files 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 identifier 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 lmfit 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 significant. 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 identified 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 identified in the JFB organoids were compared to a comprehensive list of ISGs 53 using a Python script. Declarations Author Contributions MH: Conceptualization, Methodology, Investigation, Formal Analysis, Writing – original draft; TAS: Methodology, Investigation; JFH: Investigation, Methodology, Formal Analysis; D. Snyder: Investigation; KNL: Investigation, Methodology; SDB: Investigation, Methodology, Formal Analysis; SGM: Investigation, Methodology; MDC: Investigation, Methodology; D. Skwarchuk: Investigation; DC: Methodology; AR: Investigation; BS: Investigation, Visualization; AK: Methodology, Investigation, Supervision; EKL: Resources, Methodology; MPT: Resources, Methodology, Supervision; CC: Conceptualization, Funding acquisition, Supervision; JNW: Conceptualization, Funding Acquisition; SW: Conceptualization, Funding Acquisition; TS: Resources; MJ: Conceptualization, Funding acquisition, Supervision; DB: Conceptualization, Funding acquisition, Project administration, Formal Analysis, Writing – original draft, Supervision. All authors have reviewed the final draft of the manuscript. Acknowledgements Funding from the National Institutes of Health (U01EB029242-02S1, DB, CBC, MAJ, STW, JNW; R01AI140442, TS), the Montana State University Office of Research, Economic Development and Graduate Education (DB, CBC), the Montana Agricultural Experiment Station (DB, MAJ), and the Kopriva Family Foundation (MH) is gratefully acknowledged. We greatly appreciate the support of Dr. Aga Apple, Evelyn Benson, and Caylee Falvo for help with developing bat-specific experimental methods, B. Tegner Jacobson for preparing a Python script to compare comprehensive protein lists, and Conner Killeen and Travis Van Leeuwen for performing OrganoSeg analyses. We also would like to thank Drs. Raina Plowright, Arinjay Banerjee, Vincent Munster, and Emi DeWit for helpful discussions in the design and throughout this study. The National Resource for Quantitative Proteomics is supported by NIH grant R24GM137786. Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 79 partner repository with the dataset identifier PXD036016. References Irving AT, Ahn M, Goh G, Anderson DE, Wang L-F. Lessons from the host defences of bats, a unique viral reservoir. Nature 589 , 363–370 (2021). Hoffmann M, et al. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PloS one 8 , e72942-e72942 (2013). Olival KJ, Hayman DTS. Filoviruses in bats: current knowledge and future directions. Viruses 6 , 1759–1788 (2014). Memish ZA, et al. Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg Infect Dis 19 , 1819–1823 (2013). Leroy EM, et al. Fruit bats as reservoirs of Ebola virus. Nature 438 , 575–576 (2005). Munster VJ, et al. Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis). Sci Rep 6 , 21878 (2016). Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579 , 270–273 (2020). Mallapaty S. Closest known relatives of virus behind COVID-19 found in Laos. Nature 597 , 603 (2021). Schountz T. Immunology of bats and their viruses: challenges and opportunities. Viruses 6 , 4880–4901 (2014). Watanabe S, et al. Bat coronaviruses and experimental infection of bats, the Philippines. Emerg Infect Dis 16 , 1217–1223 (2010). Subudhi S, et al. A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat. J Gen Virol 98 , 2297–2309 (2017). Tong S, et al. New world bats harbor diverse influenza A viruses. PLoS Pathog 9 , e1003657 (2013). Carding SR, Davis N, Hoyles L. Review article: the human intestinal virome in health and disease. Aliment Pharmacol Ther 46 , 800–815 (2017). Vemuri R, Shankar EM, Chieppa M, Eri R, Kavanagh K. Beyond Just Bacteria: Functional Biomes in the Gut Ecosystem Including Virome, Mycobiome, Archaeome and Helminths. Microorganisms 8 , (2020). Muñoz-Fontela C, et al. Animal models for COVID-19. Nature 586 , 509–515 (2020). Banerjee A, Misra V, Schountz T, Baker ML. Tools to study pathogen-host interactions in bats. Virus Res 248 , 5–12 (2018). Clevers H. Modeling Development and Disease with Organoids. Cell 165 , 1586–1597 (2016). Saxena K, et al. Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology. J Virol 90 , 43–56 (2016). de Souza N. Organoids. Nature Methods 15 , 23–23 (2018). Rookmaaker MB, Schutgens F, Verhaar MC, Clevers H. Development and application of human adult stem or progenitor cell organoids. Nature Reviews Nephrology 11 , 546 (2015). Blutt SE, Crawford SE, Ramani S, Zou WY, Estes MK. Engineered Human Gastrointestinal Cultures to Study the Microbiome and Infectious Diseases. Cell Mol Gastroenterol Hepatol 5 , 241–251 (2018). Powell RH, Behnke MS. WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals. Biol Open 6 , 698–705 (2017). Elbadawy M, et al. Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus. Int J Mol Sci 22 , (2021). Lei X, et al. Activation and evasion of type I interferon responses by SARS-CoV-2. Nature Communications 11 , 3810 (2020). Zhou J, et al. Infection of bat and human intestinal organoids by SARS-CoV-2. Nature Medicine 26 , 1077–1083 (2020). Clayton E, Munir M. Fundamental Characteristics of Bat Interferon Systems. Front Cell Infect Microbiol 10 , 527921 (2020). De La Cruz-Rivera PC, et al. The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. J Immunol 200 , 209–217 (2018). Subudhi S, Rapin N, Misra V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. Viruses 11 , (2019). Irving AT, et al. Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection. Cell Rep 33 , 108345 (2020). Sarkis S, et al. A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR. Dev Comp Immunol 85 , 95–107 (2018). Xie J, et al. Dampened STING-Dependent Interferon Activation in Bats. Cell Host Microbe 23 , 297–301 e294 (2018). Franz KM, Neidermyer WJ, Tan YJ, Whelan SPJ, Kagan JC. STING-dependent translation inhibition restricts RNA virus replication. Proc Natl Acad Sci U S A 115 , E2058-E2067 (2018). Machain-Williams C, et al. Serologic evidence of flavivirus infection in bats in the Yucatan Peninsula of Mexico. J Wildl Dis 49 , 684–689 (2013). Strumpf AA, Malmlov A, Ayers JD, Schountz T, Kendall LV. Hematologic Values of Jamaican Fruit Bats (Artibeus jamaicensis) and the Effects of Isoflurane Anesthesia. Journal of the American Association for Laboratory Animal Science: JAALAS 59 , 275–281 (2020). Gerrard DL, et al. Transcriptomic Signatures of Tacaribe Virus-Infected Jamaican Fruit Bats. mSphere 2 , (2017). Malmlov A, et al. Experimental Zika virus infection of Jamaican fruit bats (Artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells. PLoS Negl Trop Dis 13 , e0007071 (2019). Reid JE, Jackson AC. Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants. J Neurovirol 7 , 511–517 (2001). Shaw TI, et al. Transcriptome sequencing and annotation for the Jamaican fruit bat (Artibeus jamaicensis). PLoS One 7 , e48472 (2012). Wang K, Tian S, Galindo-Gonzalez J, Davalos LM, Zhang Y, Zhao H. Molecular adaptation and convergent evolution of frugivory in Old World and neotropical fruit bats. Mol Ecol 29 , 4366–4381 (2020). Miyoshi H, Stappenbeck TS. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nature protocols 8 , 2471–2482 (2013). Borten MA, Bajikar SS, Sasaki N, Clevers H, Janes KA. Automated brightfield morphometry of 3D organoid populations by OrganoSeg. Sci Rep 8 , 5319 (2018). Kip AM, et al. Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis 12 , 95 (2021). Lamers MM, et al. SARS-CoV-2 productively infects human gut enterocytes. Science 369 , 50–54 (2020). Triana S, et al. Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. Mol Syst Biol 17 , e10232 (2021). Jang KK, et al. Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection. PLoS Biol 20 , e3001592 (2022). Wolfel R, et al. Virological assessment of hospitalized patients with COVID-2019. Nature 581 , 465–469 (2020). Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. 22 , 2243–2252 (2008). Stanifer ML, et al. Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells. Cell Rep 32 , 107863 (2020). Stanifer ML, Guo C, Doldan P, Boulant S. Importance of Type I and III Interferons at Respiratory and Intestinal Barrier Surfaces. Front Immunol 11 , 608645 (2020). Zhou P, et al. Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity. J Immunol 186 , 3138–3147 (2011). Searle BC, et al. Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun 9 , 5128 (2018). Pruitt KD, et al. RefSeq: an update on mammalian reference sequences. Nucleic Acids Res 42 , D756-763 (2014). OhAinle M, et al. A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV. Elife 7 , (2018). Chen EY, et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14 , 128 (2013). Wang X, Zhang L, Li P, Zheng Y, Yang Y, Ji S. Apelin/APJ system in inflammation. Int Immunopharmacol 109 , 108822 (2022). Bouhaddou M, et al. The Global Phosphorylation Landscape of SARS-CoV-2 Infection. Cell 182 , 685–712 e619 (2020). Riva L, et al. Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing. Nature 586 , 113–119 (2020). Baggen J, et al. Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2. Nat Genet 53 , 435–444 (2021). Gamage AM, et al. Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles. PLoS Pathog 16 , e1009130 (2020). DeDiego ML, et al. Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis. PLoS Pathog 7 , e1002315 (2011). Schlottau K, et al. SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study. Lancet Microbe 1 , e218-e225 (2020). Hall JS, et al. Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2. Transbound Emerg Dis 68 , 3443–3452 (2021). Lin WW, Tsay AJ, Lalime EN, Pekosz A, Griffin DE. Primary differentiated respiratory epithelial cells respond to apical measles virus infection by shedding multinucleated giant cells. Proc Natl Acad Sci U S A 118 , (2021). Aicher SM, et al. Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells. J Virol 96 , e0060822 (2022). Yan H, et al. ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species. Nat Ecol Evol 5 , 600–608 (2021). Johnson BA, et al. Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. Nature 591 , 293–299 (2021). Schountz T, Baker ML, Butler J, Munster V. Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans. Front Immunol 8 , 1098 (2017). Zang R, et al. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol 5 , (2020). Brook CE, et al. Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence. Elife 9 , (2020). Mosbauer K, et al. The Effect of Allicin on the Proteome of SARS-CoV-2 Infected Calu-3 Cells. Front Microbiol 12 , 746795 (2021). Vidova V, Spacil Z. A review on mass spectrometry-based quantitative proteomics: Targeted and data independent acquisition. Anal Chim Acta 964 , 7–23 (2017). Tsai YH, et al. A Method for Cryogenic Preservation of Human Biopsy Specimens and Subsequent Organoid Culture. Cell Mol Gastroenterol Hepatol 6 , 218–222 e217 (2018). Sebrell TA, et al. Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events. Cell Tissue Res 371 , 293–307 (2018). Sebrell TA, et al. A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium. Cell Mol Gastroenterol Hepatol 8 , 157–171 e153 (2019). Hedges JF, et al. An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model. Front Immunol 13 , 918881 (2022). Loveday EK, et al. Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure. Viruses 13 , (2021). Ridley RG. Antibodies: A Laboratory Manual , 2009/04/14 edn. Cambridge University Press (1989). Gessulat S, et al. Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning. Nat Methods 16 , 509–518 (2019). Perez-Riverol Y, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res 47 , D442-D450 (2019). Graw S, et al. proteiNorm - A User-Friendly Tool for Normalization and Analysis of TMT and Label-Free Protein Quantification. ACS Omega 5 , 25625–25633 (2020). Ritchie ME, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43 , e47 (2015). Kuleshov MV, et al. The COVID-19 Drug and Gene Set Library. Patterns (N Y) 1 , 100090 (2020). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaltable1upanddownregulatedproteins.xlsx Supplemental Table 1 Supplementaltable2ISGs.xlsx Supplemental Table 2 FINALSupplementalmaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Diane","middleName":"","lastName":"Bimczok","suffix":""}],"badges":[],"createdAt":"2022-12-03 16:20:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2340919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2340919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-023-42610-x","type":"published","date":"2023-10-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30222816,"identity":"a5e5407e-7aa4-49f0-b023-8477b8226064","added_by":"auto","created_at":"2022-12-12 18:43:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":809724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment and culture of gastrointestinal organoids from Jamaican fruit bats. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Organoid derivation from Jamaican fruit bat (JFB) distal small intestine. Tissue of origin, isolated intestinal crypts and formed organoids are shown. Scale bar: 200 µm for distal SI, others are 50 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Morphology of distal SI tissue (left) and distal small intestinal organoids (right). Formalin-fixed, paraffin-embedded sections were stained with H\u0026amp;E (top row) or Alcian Blue (bottom row). High magnification insets show columnar cell shape and morphology of mucus-secreting goblet cells. Bars: 100 µm. (\u003cstrong\u003eC\u003c/strong\u003e) Size and morphology of distal SI organoids were analyzed over six consecutive passages using OrganoSeg \u003csup\u003e41\u003c/sup\u003e. Dots: individual organoids (n≥6); bars: mean ± SD.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/7fef418740f13a0de5055375.png"},{"id":30223220,"identity":"3a99c5c5-42a3-4261-bdc0-26fc4c875701","added_by":"auto","created_at":"2022-12-12 18:51:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":396858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of gastrointestinal organoids from Jamaican fruit bats.\u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Conservation of tissue-specific gene expression patterns in JFB organoids derived from stomach, proximal and distal SI. Pooled qRT-PCR data from n=4 established organoid lines (p2-10) are shown; mean ± SEM. Statistical analyses were performed using ANOVA with Tukey’s multiple comparisons test; *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01. (\u003cstrong\u003eB\u003c/strong\u003e) Expression of select intestinal epithelial cell-specific proteins. JFB distal SI organoids (3 technical replicates) were lysed and processed for data-independent acquisition mass spectrometry. Individual datapoints and mean ± SD. (\u003cstrong\u003eC\u003c/strong\u003e) Barrier function of JFB organoid cells cultured on transwell inserts for 10 days. Representative data of distal small intestinal (SI) organoids with mean ± SEM of triplicate wells (left) and pooled data from four experiments (right) with gastric, proximal SI and distal SI organoids are shown. (\u003cstrong\u003eD\u003c/strong\u003e) Confocal imaging reveals polarized expression of cytokeratin in JFB organoids. Left: brightfield image, right: single Z-plane; pan-cytokeratin-red, nuclei-blue. One representative of 3 experiments. Bars: 25 µm.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/935c7f894e84932b08012e77.png"},{"id":30222819,"identity":"33171561-e7e5-4b3d-b338-0102109baa37","added_by":"auto","created_at":"2022-12-12 18:43:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":751396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReplication of SARS-CoV-2 in JFB intestinal organoids. \u003c/strong\u003eDissociated JFB distal intestinal organoids were inoculated with SARS-CoV-2 (strain USA-WA1/2020) for 2 h or were mock-treated and then were washed and re-embedded in Matrigel. At 48 and 72 h post infection, RNA was extracted from (\u003cstrong\u003eA\u003c/strong\u003e) the organoids and (\u003cstrong\u003eB\u003c/strong\u003e) the culture supernatants, and replication of SARS-CoV-2 was analyzed by quantitative real-time PCR (qRT-PCR) for the envelope (E) gene using the standard curve method. (\u003cstrong\u003eC\u003c/strong\u003e) RNA extracted from the organoids was analyzed for viral sgRNA (E gene) using a leader-specific primer. (\u003cstrong\u003eD\u003c/strong\u003e) Supernatants from SARS-CoV-2 infected organoids or Vero E6 cells were analyzed by plaque assay for the presence of infectious SARS-CoV-2. (A-D) Panels show data from one representative out of three to four independent experiments with two or three technical replicates as mean ± SEM, analyzed by ANOVA with Dunnett’s or Tukey’s multiple comparisons test; *P≤0.05, **P≤0.01, ***P≤0.0001. (\u003cstrong\u003eE\u003c/strong\u003e) SARS-CoV-2 protein detection in isolated epithelial cells, but not in intact JFB intestinal organoids. Organoids or Vero E6 cells were fixed and permeabilized at 48 h post SARS-CoV-2 infection (MOI 10) and then were stained with DAPI (blue), phalloidin (green) and a monoclonal antibody to SARS-CoV-2 spike protein (red). Arrows point out cells containing SARS-CoV-2 spike protein. Data are representative of three independent experiments. Scale bar= 25 μm.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/54542779bcf842d4cdce8e0d.png"},{"id":30221926,"identity":"56ecc9d1-3723-4913-bc32-e8d8bb9b9e4a","added_by":"auto","created_at":"2022-12-12 18:35:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":746197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased growth of JFB organoids infected with SARS-CoV-2.\u003c/strong\u003eDissociated JFB distal intestinal organoids or Vero E6 cells were mock-inoculated or were infected with SARS-CoV-2 at an MOI of 1 or 10, as described above, with NucView® 488, a cell membrane-permeable fluorogenic caspase-3 reporter, added to the medium. (\u003cstrong\u003eA\u003c/strong\u003e) At 48 h post infection, the cells were imaged using fluorescence and phase contrast (brightfield) microscopy. Scale bars = 50 μm. (\u003cstrong\u003eB\u003c/strong\u003e) ImageJ was used to quantitate NucView® fluorescence based on pixel counts in thresholded digital images of manually selected organoids. Individual data points, mean ± SD of one representative of four independent experiments with three technical replicates is shown, data were analyzed by Student’s \u003cem\u003et \u003c/em\u003etest. (\u003cstrong\u003eC\u003c/strong\u003e) Organoid size in SARS-CoV-2-infected organoid cultures after 48 h was determined on brightfield images using ImageJ. Individual data points, mean ± SD of one representative of five independent experiments with three technical replicates is shown, data were analyzed by ANOVA with Tukey’s multiple comparisons test; **\u003cem\u003eP\u003c/em\u003e≤0.01. (\u003cstrong\u003eD\u003c/strong\u003e) Number of detected organoids in random brightfield images from mock-infected and SARS-CoV-2 infected JFB organoid cultures (MOI 10, 48 h). Pooled data from four independent experiments; Student’s \u003cem\u003et \u003c/em\u003etest, *\u003cem\u003eP\u003c/em\u003e≤0.05.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/2bd5296abbd9f78542955e8b.png"},{"id":30223221,"identity":"e93dc0c2-dfd9-4e8d-adfa-3c328565a98b","added_by":"auto","created_at":"2022-12-12 18:51:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":365474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJFB distal intestinal organoids express antiviral and pro-inflammatory genes in response to infection with SARS-CoV-2. (A-D)\u003c/strong\u003e Dissociated JFB distal intestinal organoids were infected with active SARS-CoV-2 at an MOI of 1 or 10. The unbound virus was washed off, and the cells were re-plated in Matrigel. After 48 or 72 h, the RNA was extracted from the cells to evaluate genes expression via quantitative real-time PCR (qRT-PCR). Data from one representative out of four independent experiments run in triplicate are shown as mean ± SEM. (\u003cstrong\u003eE-H):\u003c/strong\u003e Organoids were treated with UV-inactivated SARS-CoV-2 at 10 µg/mL, or with a panel of TLR agonists (TLR2: heat-killed \u003cem\u003eL. monocytogenes, \u003c/em\u003eHKLM; TLR3: low MW poly I:C; TLR7: imiquimod, TLR9: ODN2006) and then were analyzed by qRT-PCR 48 h after stimulation. Pooled data for three independent experiments; mean ± SEM are shown. Graphs show gene expression of (\u003cstrong\u003eA, E\u003c/strong\u003e) \u003cem\u003eIfna4l \u003c/em\u003e(IFNα 4-like), (\u003cstrong\u003eB, F\u003c/strong\u003e) \u003cem\u003eIfnb \u003c/em\u003e(IFN-β), (\u003cstrong\u003eC, G\u003c/strong\u003e) \u003cem\u003eIl6 \u003c/em\u003e(IL-6) and (\u003cstrong\u003eD, H\u003c/strong\u003e) \u003cem\u003etnf \u003c/em\u003e(TNF-α\u003cem\u003e). \u003c/em\u003eAll data were analyzed using the 2\u003csup\u003e(-ΔΔCt)\u003c/sup\u003e method with \u003cem\u003egapdh\u003c/em\u003e as a housekeeping gene and are expressed as fold change relative to the mock-infected control. ANOVA with Dunnett’s multiple comparisons test; *\u003cem\u003eP\u003c/em\u003e≤0.05, **\u003cem\u003eP\u003c/em\u003e≤0.01, ***\u003cem\u003eP\u003c/em\u003e≤0.0001.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/9cdf62141a2eaf79cd01a8ce.png"},{"id":30223953,"identity":"89e118c5-d9f0-4907-9332-4efd1a7e5740","added_by":"auto","created_at":"2022-12-12 18:59:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1093950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteome analysis of SARS-CoV-2-infected JFB organoids at 48 h. \u003c/strong\u003eJFB distal SI organoids were infected with SARS-CoV-2 at an MOI of 10 or underwent mock treatment for 48 h and then were lysed and processed for data-independent acquisition (DIA) mass spectrometry.\u003cstrong\u003e \u003c/strong\u003eN=3 replicates from one organoid line were analyzed. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eVolcano plot showing all detected proteins and protein isoforms. Proteins with significantly increased or decreased expression (≥2-fold change; \u003cem\u003eP\u003c/em\u003e≤0.05) are shown in red and blue. (\u003cstrong\u003eB\u003c/strong\u003e) Expression of interferon-stimulated genes (ISGs), identified based on OhAinle et al. (2018) \u003csup\u003e53\u003c/sup\u003e, in mock-infected and SARS-CoV-2 infected JFB organoids. Proteins with significantly increased or decreased expression (≥2-fold change;\u003cem\u003e P\u003c/em\u003e≤0.05) are shown in red and blue. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap showing relative change (Z-scores) of all 27 significantly upregulated proteins and of the top 30 downregulated proteins (\u003cem\u003eP\u003c/em\u003e≤0.05). Data from triplicate cultures are shown. Protein function was determined using UniProtKB (\u003cem\u003eH. sapiens).\u003c/em\u003e Significantly regulated ISGs are highlighted in yellow. (\u003cstrong\u003eD\u003c/strong\u003e) IPA analysis showing top regulated canonical signaling pathways (top) and molecular and cellular functions (bottom) activated in SARS-CoV-2 infected JFB organoids. (\u003cstrong\u003eE,F\u003c/strong\u003e) Enrichr pathway analysis using (\u003cstrong\u003eE\u003c/strong\u003e) the 2021 human KEGG pathway database and (\u003cstrong\u003eF\u003c/strong\u003e) the 2021 COVID-19 related gene sets. Pathways were ranked based on combined score ranking.\u003c/p\u003e","description":"","filename":"NCBatorganoidFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/a0a29495f4d29c01722cd361.png"},{"id":45394794,"identity":"b416ad97-6154-42b5-88e8-ff827e5654f2","added_by":"auto","created_at":"2023-10-29 07:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2598838,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/abf59a94-0cb1-4182-96fd-12b42d350919.pdf"},{"id":30223219,"identity":"34aa77f5-308e-4daa-a86b-11e348f54214","added_by":"auto","created_at":"2022-12-12 18:51:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":91469,"visible":true,"origin":"","legend":"Supplemental Table 1","description":"","filename":"Supplementaltable1upanddownregulatedproteins.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/08e66b829859f8467222dff7.xlsx"},{"id":30221923,"identity":"2b64b9ad-1a2f-401b-935f-292f3ef5ddae","added_by":"auto","created_at":"2022-12-12 18:35:55","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":48837,"visible":true,"origin":"","legend":"Supplemental Table 2","description":"","filename":"Supplementaltable2ISGs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/a98861bdbcee02733639e832.xlsx"},{"id":30221930,"identity":"1f434449-2765-4b16-b0d1-940bd208dad1","added_by":"auto","created_at":"2022-12-12 18:35:55","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":361080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"FINALSupplementalmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2340919/v1/7bffb111cb25429e84b43971.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBats are considered important natural reservoirs for a variety of emerging zoonotic viruses that cause several illnesses in humans and other mammals \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Hendra virus, Ebola virus, and Marburg virus \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The ongoing COVID-19 pandemic is caused by severe acute respiratory coronavirus-2 (SARS-CoV-2) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, 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 \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e) and BANAL-52 (96.8% identity \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e), 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 \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In a study by Watanabe \u003cem\u003eet al.\u003c/em\u003e on wild bats captured in the Philippines \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, enteric coronaviruses were detected in \u0026gt;\u0026thinsp;50% of the animals, but clinical signs of disease were absent. Similarly, Subudhi \u003cem\u003eet al.\u003c/em\u003e found that 30% of North American little brown bats harbored coronaviruses in their intestines but did not display any signs of illness \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Tong \u003cem\u003eet al.\u003c/em\u003e analyzed rectal swabs and intestinal tissues from asymptomatic fruit bats in Peru and identified a novel influenza A virus, H18N11 \u003csup\u003e12\u003c/sup\u003e. In contrast to bats, where gastrointestinal infections with eukaryotic viruses are frequent and are commonly asymptomatic \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, a similar colonization of the human gut with non-pathogenic eukaryotic viruses has not been reported, pointing to species-specific mechanisms \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudying coronavirus infection in the GI tracts of bats is difficult, since few institutions maintain bat colonies for \u003cem\u003ein vivo\u003c/em\u003e infection experiments, and cell lines from the GI tract of bats are not available, limiting \u003cem\u003ein vitro\u003c/em\u003e analyses \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Organoid cultures have untapped potential as a model to study the mechanisms of viral infection in bat cells \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. 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 \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Importantly, growth conditions for GI organoids appear similar across multiple species \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Two previous studies have described the generation of intestinal organoid cultures from bat species \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Intestinal organoids developed from Chinese horseshoe bats, \u003cem\u003eRhinolophus sinicus\u003c/em\u003e, showed susceptibility to SARS-CoV-2, but lacked long-term active proliferation past 4\u0026ndash;5 weeks \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Intestinal organoids derived from Leschenault\u0026rsquo;s rousette, \u003cem\u003eRousettus leschenaultii\u003c/em\u003e. showed susceptibility to \u003cem\u003ePteropine\u003c/em\u003e orthoreovirus, but not SARS-CoV-2 \u003csup\u003e23\u003c/sup\u003e. However, neither of these studies evaluated the cellular antiviral mechanisms of bat organoid tissues \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe hypothesis that altered IFN responses in bats compared to other species promote increased viral tolerance is a central paradigm in bat immunology \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In Australian black flying foxes (\u003cem\u003eP. alecto)\u003c/em\u003e, a high level of constitutive IFN-α expression was detected, which has led to the concept that an \u0026ldquo;always on\u0026rdquo; IFN signaling system in bats can effectively suppress viral replication and prevent disease early on after infection \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e29\u003c/sup\u003e and the ISG oligoadenylate synthase 1 (OAS1) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Importantly, these characteristics of the IFN system appear to be unique to particular bat species, pointing to a need for more detailed analyses.\u003c/p\u003e \u003cp\u003eJamaican 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 \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. JFBs also are susceptible to experimental infection with Zika virus and MERS-CoV \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Based on the recently annotated genome \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, JFBs have one interferon (IFN)-β gene, five IFN-α genes, and five IFN-ω genes. Multiple interferon regulatory factors (IRFs) have also been identified, making JFBs a tractable model system for studies of antiviral immunity.\u003c/p\u003e \u003cp\u003eHere 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 \u0026ldquo;always on\u0026rdquo; paradigm for antiviral interferon responses in bats \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, the organoids mounted a robust interferon response to infection with active SARS-CoV-2. Proteomics and pathway analysis revealed that the JFB organoid proteome profiles matched profiles found in other SARS-CoV-2 infection studies and that SARS-CoV-2 infection activated innate inflammatory and cellular repair responses in this model system.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDevelopment and Characterization of JFB Gastrointestinal Organoids.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe established JFB gastrointestinal organoid cultures from fresh and cryopreserved stomach and from proximal and distal small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cb\u003eSupplemental Fig.\u0026nbsp;1A, B\u003c/b\u003e). 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 (\u003cb\u003eSupplemental Fig.\u0026nbsp;1C\u003c/b\u003e).The murine noggin, R-spondin, and Wnt3a secreted by the L-WRN cells \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e show protein sequence similarities of 98%, 86%, and 99% with the orthologous JFB proteins, suggesting that these factors would be active in JFB cells (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eEstablished JFB organoids mimicked the epithelial structure of JFB gastrointestinal tissue, with a simple columnar epithelium, a basal nucleus and a defined luminal space (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eSupplemental Fig.\u0026nbsp;3A, B\u003c/b\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eSupplemental Fig.\u0026nbsp;3A, B\u003c/b\u003e). Morphometric analysis with OrganoSeg \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e showed that organoid size varied between different passages, but with no clear trends, and organoid shape also did not change significantly over six consecutive passages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). JFB organoids were maintained for at least 30 passages (\u0026gt;\u0026thinsp;6 months), and also were successfully cryopreserved and re-cultured from cryopreserved stocks (data not shown).\u003c/p\u003e \u003cp\u003eWe next performed transcriptional analysis of the organoids to confirm tissue-specific gene expression patterns. The distal and proximal intestinal organoids expressed the intestine-specific genes \u003cem\u003eVil1, Cdx2\u003c/em\u003e, and \u003cem\u003eMuc2\u003c/em\u003e, while the gastric organoids showed increased expression of the chief cell marker pepsinogen C (\u003cem\u003ePgc\u003c/em\u003e) with low expression of \u003cem\u003eVil1\u003c/em\u003e, \u003cem\u003eCdx2\u003c/em\u003e and \u003cem\u003eMuc2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Since no specific 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\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ATPase, claudin 18, and a mucin (MUC5AC-like) were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, confirming 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Confocal imaging analysis of cytokeratin expression confirmed epithelial cell polarization and correct inside-in orientation of the organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these analyses demonstrate that gastrointestinal organoids from JFBs replicate key features of the gastrointestinal epithelium.\u003c/p\u003e\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eInfection of JFB distal organoids with SARS-CoV-2 leads to replication of viral genomes.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTo 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 \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Quantitative PCR analysis of viral genomes in JFB organoid cell lysates revealed a significant, concentration-dependent increase (\u0026gt;\u0026thinsp;1 log, \u003cem\u003eP\u0026thinsp;\u0026le;\u003c/em\u003e\u0026thinsp;0.05) in SARS-CoV-2 gene E RNA at 48 and 72 hours post infection (hpi, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The SARS-CoV-2 PCR in culture supernatants showed a similar increase at an MOI of 1 at 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Importantly, significant expression of subgenomic (sg)RNA (\u0026gt;\u0026thinsp;2 log-fold above baseline) for gene E indicating active viral replication in the organoids also was identified \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, albeit at low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). 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 \u0026gt;\u0026thinsp;1 log-fold, suggesting a loss of infectivity over time (\u003cb\u003eSupplemental Fig.\u0026nbsp;4\u003c/b\u003e). Interestingly, immunofluorescence 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLack of cytopathic effect but increased growth in SARS-CoV-2 infected JFB organoids\u003c/h2\u003e \u003cp\u003eWe also evaluated the cell viability of JFB distal intestinal organoids following SARS-CoV-2 infection by measuring caspase-3 activity with NucView\u0026reg; \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). 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 \u003cem\u003eRhinolophus sinicus\u003c/em\u003e organoids\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, SARS-CoV-2 did not appear to have a cytopathic effect in JFB organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B), since the proportion of apoptotic cells did not change upon infection. Interestingly, SARS-CoV-2 caused a significant increase in organoid size and in the number of organoids that had re-formed from single cells after 48 h of infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D), indicating that viral infection triggered increased cell proliferation in the bat intestinal epithelium.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSARS-CoV-2 induces expression of type I interferons and proinflammatory cytokines in JFB organoids.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eUnique characteristics of the interferon (IFN) system have been linked to the increased viral tolerance observed in many bat species \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Therefore, we used quantitative RT-PCR to analyze gene expression of type I interferons and proinflammatory cytokines in JFB distal small intestinal organoids following 48 h exposure to SARS-CoV-2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, expression of type I interferon \u003cem\u003eIfna4l\u003c/em\u003e was upregulated at 48 hpi with an MOI of 10, while an MOI of 1 caused significant upregulation of the gene at 72 hpi. Gene expression of \u003cem\u003eIfnb\u003c/em\u003e also was significantly increased with both MOIs at 48 hpi and remained elevated with the lower viral dose at 72 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Type III IFNs are known to play a role in mucosal antiviral immunity and SARS-CoV-2 infection and also may have unique functions in bats \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, the type III IFN loci in JFBs are poorly annotated \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and we unable to generate functional primers based on the published genome. Interestingly, organoid infection with SARS-CoV-2 at an MOI of 1 significantly increased expression of the proinflammatory cytokines \u003cem\u003eTnf\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e at 48 hpi, and \u003cem\u003eIl6\u003c/em\u003e remained elevated at 72 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D). The above data suggest that JFB distal organoids exhibited an anti-viral and pro-inflammatory response to SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003eTo determine whether active viral infection was responsible for the observed induction of antiviral and inflammatory genes, or whether gene expression was induced by unspecific 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 inflammatory cytokines 6 h post inoculation (\u003cb\u003eSupplemental Fig.\u0026nbsp;5\u003c/b\u003e). However, no significant upregulation of these genes was observed with any of the stimuli at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-H). These observations suggest that active infection with SARS-CoV-2 is required for sustained upregulation of antiviral and inflammatory gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImpact of SARS-CoV-2 infection on the JFB intestinal epithelial cell proteome\u003c/h2\u003e \u003cp\u003eA quantitative proteomic workflow 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 files retrieved from the \u003cem\u003eA. jamaicensis\u003c/em\u003e reference genome \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. 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\u0026thinsp;\u0026ge;\u0026thinsp;2-fold change at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA \u003cb\u003eand Supplemental Table\u0026nbsp;1\u003c/b\u003e). To better understand antiviral responses in the JFB intestine, we next compared the identified proteins to a comprehensive list of human interferon-stimulated genes (ISGs, \u003cb\u003eSupplemental Table\u0026nbsp;2\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Interestingly, 100 of all identified JFB proteins could tentatively be classified as ISGs. However, only one of the ISG proteins, ribonucleases P/MRP protein subunit POP1 (POP1), was significantly upregulated in response to SARS-CoV-2, while four ISG proteins (ERLEC1, CFB, ARMCX3 and ITIH2) were significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Several of these proteins are involved in regulation of cell turnover and posttranslational modifications. We next performed Ingenuity Pathway Analysis (IPA) and Enrichr analysis \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e 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 significantly regulated pathway, followed by Apelin liver signaling \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, which is involved in intestinal inflammation, repair, and wound healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Top regulated cellular functions were cell assembly, organization, maintenance, movement, signaling and morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings suggest that SARS-CoV-2 triggers regenerative response pathways, consistent with the increased organoid size observed in the SARS-CoV-2-infected compared to mock-infected cultures. Similarly, Enrichr identified significant 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Importantly, Enrichr analysis also found multiple significant matches for protein signatures that were previously found to be upregulated in SARS-CoV-2 infection in various experimental systems \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Overall, the proteomics analysis points to the activation of innate inflammatory and regenerative pathways along with characteristic COVID-19 signatures upon SARS-CoV-2 infection of the JFB intestinal epithelium.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established and characterized organoid cultures from the proximal and distal small intestine and stomach of JFBs. Using this model, we investigated the response of JFB intestinal epithelial cells to infection with SARS-CoV-2. Considering that JFBs are susceptible to MERS-CoV, Zika virus, and rabies virus \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, we evaluated the susceptibility of the JFB distal intestinal organoids to SARS-CoV-2. Notably, JFBs are not thought to be natural carriers of SARS-CoV-2, and no studies on \u003cem\u003ein vivo\u003c/em\u003e infection of JFBs with SARS-CoV-2 have been published to date. Considering the vast number and associated genetic diversity of bat species it is not surprising that SARS-CoV-2 infection experiments in other bat species have yielded conflicting results. In Egyptian fruit bats (\u003cem\u003eRousettus aegyptiacus)\u003c/em\u003e, transient asymptomatic respiratory tract infection with viral replication in lung and trachea and oral and fecal shedding was achieved upon experimental SARS-CoV-2 inoculation \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Conversely, American big brown bats (\u003cem\u003eEptesicus fuscus\u003c/em\u003e) appeared resistant to infection with SARS-CoV-2 \u003csup\u003e62\u003c/sup\u003e. Likewise, intestinal organoids derived from two different bat species responded differently to SARS-CoV-2 infection. Organoids from Chinese horseshoe bats, where SARS-CoV-2-like virus has been detected \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, produced infectious SARS-CoV-2 virions at similar levels as human intestinal organoids \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In contrast, intestinal organoids from Leschenault's rousette bats (\u003cem\u003eRousettus leschenaultii\u003c/em\u003e) failed to support SARS-CoV-2 replication \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Interestingly, PCR analysis revealed a significant increase in viral and sgRNA in the JFB distal organoids at 48 and 72 hpi, which demonstrates initiation of viral replication in the organoids. SARS-CoV-2 genomes also were significantly increased in organoid culture supernatants. Using immunohistochemistry, we detected SARS-CoV-2 spike protein in individual cells, but not in morphologically intact JFB organoids. This observation may reflect shedding of viable virus-infected cells from the epithelial monolayer, as described for other viral infections \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We also did not detect infectious virions in organoid cells or supernatants using plaque assays in VeroE6 cells, suggesting that JFB intestinal organoids support incomplete SARS-CoV-2 infection. A similar limited and incomplete replication of SARS-CoV-2 was also reported in cell lines from several different bat species, even after transduction with human ACE2, in a recent study by Aicher \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. However, the presence of sgRNA and of SARS-CoV-2 protein in some cells suggest that entry and replication of the virus did occur in the JFB organoids. This interpretation is consistent with a study by Yan \u003cem\u003eet al.\u003c/em\u003e that predicted a moderate ability of SARS-CoV-2 to infect JFB cells based on the protein sequence of the SARS-CoV-2 receptor ACE2 \u003csup\u003e65\u003c/sup\u003e and our unpublished observations of ACE-2 gene expression on the JFB organoids. Loss of the furin cleavage site in the WA01 reference stain of SARS-CoV-2 also may have had an impact on the efficacy of infection \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Further experiments are needed to evaluate at which stage of viral replication cycle SARS-CoV-2 replication stalls in the JFB organoid model and whether JFBs are permissive to SARS-CoV-2 infection \u003cem\u003ein vivo\u003c/em\u003e. Notably, many previous studies on viral infection in bats have relied solely on viral nucleic acids to measure infection \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Therefore, it is difficult to assess whether the failure to detect replication-competent virions was unique to our infection model.\u003c/p\u003e \u003cp\u003eOur results demonstrate that active SARS-CoV-2 virus induced a robust anti-viral immune response, with increased expression of IFN-α and IFN-β at 48 h after SARS-CoV-2 infection. This strong induction of interferons in response to viral infection was surprising, since the current paradigm is that the interferon system in bats is constitutively active, based on studies in Australian black flying foxes (\u003cem\u003eP. alecto)\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Conversely, potent interferon responses were detected in serotine bats (\u003cem\u003eEptesicus serotinus\u003c/em\u003e) and David\u0026rsquo;s myotis bat cells upon SARS-CoV-2 infection \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. These observed differences point to species-specific immune system characteristics in bats, consistent with the high level of genetic diversity in the order Chiroptera, which comprises over 1,400 species.\u003c/p\u003e \u003cp\u003eThe lack of a cytopathic effect in SARS-CoV-2-infected JFB organoids was an intriguing observation. It has been shown that SARS-CoV-2 causes neither apoptotic nor necrotic cell death in the gastrointestinal tract of infected human patients. Whether SARS-CoV-2 impacts viability of organoid cultures is still a matter of debate. Lamers \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e observed increased apoptotic cell death in human enteroids at 60 hpi, and Zhou \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e state that both human and horseshoe bat enteroids developed a cytopathic effect after SARS-CoV-2 inoculation. Conversely, studies by Stanifer \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and Zang \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e did not describe increased cell death in human SARS-CoV-2 infected enteroids. Data from several studies in bat cells suggest that heightened IFN responses in these cultures may prolong viral infection by limiting pathogen-induced cell death through induction of anti-apoptotic genes including BCL-2 and PMAIP1 \u003csup\u003e35, 69\u003c/sup\u003e. 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 significant 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 findings 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.\u003c/p\u003e \u003cp\u003eWe 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 first, to our knowledge, to use a DIA-based proteomics approach with JFB cells. Our analysis confirmed 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-inflammatory cytokines in SARS-CoV-2-infected JFB organoids that we detected, inflammatory 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 significant 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 \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, and may reflect a JFB-specific 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 identified by other studies on SARS-CoV-2 infection. Notably, there are several limitations to the proteomics approach undertaken in our study. First, the non-targeted DIA approach may not be sensitive enough to identify strongly regulated targets with a low overall expression level \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Second, an annotated proteome of the JFB is currently not available and thus had to be inferred from the genome, which may lead to misidentified proteins. Lastly, pathway analysis was based on human databases, which again may miss JFB-specific signaling pathways.\u003c/p\u003e \u003cp\u003eImportantly, 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 specific 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 \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and Rousettus bats \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Similar culture conditions also have been successfully used to culture intestinal organoids from cat, dog, cow, horse, pig and sheep \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. 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 tissue-specific 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.\u003c/p\u003e \u003cp\u003eTo summarize, we established and characterized JFB gastrointestinal organoids that recapitulated the organ-specific multicellular composition of JFB gastrointestinal tissue. We demonstrated SARS-CoV-2 sgRNA replication at a low efficiency 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 pro-inflammatory 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.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cb\u003eTissue samples.\u003c/b\u003e Male and female Jamaican fruit bats (\u003cem\u003eArtibeus jamaicensis)\u003c/em\u003e 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, five adult bats (4 male, 1 female) were euthanized by 5% isoflurane in O\u003csub\u003e2\u003c/sub\u003e 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).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCrypt and gland isolation methods\u003c/b\u003e. Bat tissues were processed immediately upon arrival or were cryopreserved and then thawed rapidly if needed \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. To derive organoids, proximal intestinal and distal intestinal tissues were washed in cold PBS and cut into ~\u0026thinsp;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 \u003cem\u003eg.\u003c/em\u003e 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 \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Recovered crypts/glands were resuspended in 10 \u0026micro;l of Matrigel and plated in 96-well plates. After the gel was polymerized, 200 \u0026micro;l of medium (\u003cb\u003eSupplemental Table\u0026nbsp;3\u003c/b\u003e) was added, and the plates were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for one week.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMaintenance of JFB organoids.\u003c/b\u003e 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 \u003cem\u003eg\u003c/em\u003e at 4˚C, then were resuspended Matrigel and plated in a 24-well plate. After the gel had polymerized, 500 \u0026micro;l of medium was added and the plate was incubated at 37˚C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was changed every other day and the organoids were passaged every 5\u0026ndash;7 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimization of growth conditions.\u003c/b\u003e In addition to the basic growth medium, termed L-WRN medium, described above, we also tested a commercially available growth medium, IntestiCult\u0026trade; (StemCell), a complex medium termed \u0026ldquo;colonoid medium\u0026rdquo; described by Tsai \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, and analyzed medium supplementation with a number of different growth factors commonly used in organoid culture protocols (\u003cb\u003eSupplemental Table\u0026nbsp;3\u003c/b\u003e). 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 influence 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).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological Analysis of JFB Organoid Cultures.\u003c/b\u003e Organoids were recovered from the culture plates and treated with Histogel (ThermoFisher) prior to formalin fixation and paraffin embedding, following standard protocols. Slides were stained with hematoxylin/eosin and with Alcian Blue to visualize mucus production.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSARS-CoV-2 Infection of JFB organoids.\u003c/b\u003e Bat organoids were dissociated by incubation with 350 \u0026micro;L 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\u0026deg;C with frequent gentle agitation. Notably, the SARS-CoV-2 strain used was shown to have a defective furin cleavage site \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, but readily infected inducible pluripotent stem cell-derived human intestinal organoids in control experiments. The infected bat organoids were incubated in 30 \u0026micro;L of DMEM at 37\u0026deg;C for 2 hours with occasional shaking. Organoids were collected into 500 \u0026micro;L DMEM and centrifuged at 200 \u003cem\u003eg\u003c/em\u003e for 5 min to wash. Then cells were resuspended in 30 \u0026micro;L 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\u0026deg;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 \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment of JFB organoids with TLR agonists and inactivated virus.\u003c/b\u003e To analyze transcriptional response of JFB organoids to stimulation with pathogen-associated molecular patterns, organoids were trypsinized and then re-embedded into Matrigel in the presence of the following TLR agonists (Human TLR1-9 agonist kit, InvivoGen): TLR1, Pam3CSK4, 1 \u0026micro;g/mL; TLR2, heat-killed \u003cem\u003eListeria monocytogenes\u003c/em\u003e (10\u003csup\u003e8\u003c/sup\u003e/mL); TLR3, low molecular weight poly I:C, 10 \u0026micro;g/mL; TLR7, imiquimod, 1 \u0026micro;g/mL; TLR9, ODN2006, 5 \u0026micro;M. Alternatively, organoids were treated with UV-inactivated SARS-CoV-2 \u003csup\u003e76\u003c/sup\u003e (10 \u0026micro;g/mL). After 48 h, Organoids were lysed in TRI Reagent (Sigma) and processed for RNA isolation and RT-PCR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative RT-PCR.\u003c/b\u003e 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-specific genes and cytokines were designed using NCBI primer blast using the JFB genome (\u003cem\u003eArtibeus jamaicensis\u003c/em\u003e, textid: 9417) and are listed in \u003cb\u003eSupplemental Table\u0026nbsp;4\u003c/b\u003e. GAPDH was amplified 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\u0026reg;Amp Viral RNA Mini kit (Qiagen). Viral genomes were then quantified in a single step RT-PCR reaction using primers and a TaqMan probe to the SARS-CoV-2 envelope (E) gene, as previously described \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, 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 W\u0026ouml;lfel \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. An RNA standard curve generated from a T7 \u003cem\u003ein vitro\u003c/em\u003e transcribed gBlock\u0026trade; sequence (Integrated DNA Technologies) was used for normalization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence Staining.\u003c/b\u003e For visualization of epithelial cytokeratin, we used a mouse-anti cytokeratin antibody that detects cytokeratins in a wide range of species (Thermofisher, 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 \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were immunized with 10 \u0026micro;g UV-inactivated SARS-CoV-2 (USA-WA1/2020) \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e 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 \u0026micro;g/mL. For immunofluorescence analysis, organoids were fixed 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\u0026thinsp;+\u0026thinsp;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 \u0026micro;M DAPI (MP Biomedicals, 0215757405). Actin filaments 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\u0026ndash;11 randomly selected organoids with intact morphology for each experiment and condition were recorded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability and organoid growth.\u003c/b\u003e To measure caspase 3 activity in SARS-CoV-2-infected organoids, NucView488 (Biotium) was added to the medium at 3 \u0026micro;M 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. Brightfield images of the organoid cultures were used to measure organoid size for normalization of the NucView data and for assessment of organoid growth.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProteomics analyses\u003c/strong\u003e \u003cp\u003eTriplicate 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\u0026deg; 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 profiles of the organoids. Chromatogram libraries were constructed using Prosit \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e, and proteins were identified and quantified using EncyclopeDIA, based on protein FASTA files retrieved from NCBI RefSeq for the Jamaican fruit bat (BioProject PRJNA673233) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e partner repository with the dataset identifier PXD036016. False discovery thresholds of 1% were applied. The ProteiNorm app was used to optimize data normalization \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, 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 lmfit with empirical Bayes smoothing was used for the analysis \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Proteins with an FDR-adjusted \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and an absolute fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 were considered significant. Ingenuity Pathway Analysis (Qiagen) and Enrichr \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e with combined score ranking (c\u0026thinsp;=\u0026thinsp;log(p) * z, where c\u0026thinsp;=\u0026thinsp;the combined score, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;Fisher exact test \u003cem\u003eP\u003c/em\u003e-value, and z\u0026thinsp;=\u0026thinsp;z-score) were used to identify cellular signaling pathways. COVID-19 related gene sets identified by Enrichr were based on the \u0026ldquo;The COVID-19 Drug and Gene Set Library, 2021 version\u0026rdquo; website \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. To analyze the impact of SARS-CoV-2 infection on ISGs, proteins identified in the JFB organoids were compared to a comprehensive list of ISGs \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e using a Python script.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMH: Conceptualization, Methodology, Investigation, Formal Analysis, Writing \u0026ndash; original draft; TAS: Methodology, Investigation; JFH: Investigation, Methodology, Formal Analysis; D. Snyder: Investigation; KNL: Investigation, Methodology; SDB: Investigation, Methodology, Formal Analysis; SGM: Investigation, Methodology; MDC: Investigation, Methodology; D. Skwarchuk: Investigation; DC: Methodology; AR: Investigation; BS: Investigation, Visualization; AK: Methodology, Investigation, Supervision; EKL: Resources, Methodology; MPT: Resources, Methodology, Supervision; CC: Conceptualization, Funding acquisition, Supervision; JNW: Conceptualization, Funding Acquisition; SW: Conceptualization, Funding Acquisition; TS: Resources; MJ: Conceptualization, Funding acquisition, Supervision; DB: Conceptualization, Funding acquisition, Project administration, Formal Analysis, Writing \u0026ndash; original draft, Supervision. All authors have reviewed the final draft of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding from the National Institutes of Health (U01EB029242-02S1, DB, CBC, MAJ, STW, JNW; R01AI140442, TS), the Montana State University Office of Research, Economic Development and Graduate Education (DB, CBC), the Montana Agricultural Experiment Station (DB, MAJ), and the Kopriva Family Foundation (MH) is gratefully acknowledged. We greatly appreciate the support of Dr. Aga Apple, Evelyn Benson, and Caylee Falvo for help with developing bat-specific experimental methods, B. Tegner Jacobson for preparing a Python script to compare comprehensive protein lists, and Conner Killeen and Travis Van Leeuwen for performing OrganoSeg analyses. We also would like to thank Drs. Raina Plowright, Arinjay Banerjee, Vincent Munster, and Emi DeWit for helpful discussions in the design and throughout this study. The National Resource for Quantitative Proteomics is supported by NIH grant R24GM137786.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE\u0026nbsp;\u003csup\u003e79\u003c/sup\u003e partner repository with the dataset identifier PXD036016.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIrving AT, Ahn M, Goh G, Anderson DE, Wang L-F. Lessons from the host defences of bats, a unique viral reservoir. Nature \u003cb\u003e589\u003c/b\u003e, 363\u0026ndash;370 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmann M, \u003cem\u003eet al.\u003c/em\u003e Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PloS one \u003cb\u003e8\u003c/b\u003e, e72942-e72942 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlival KJ, Hayman DTS. Filoviruses in bats: current knowledge and future directions. Viruses \u003cb\u003e6\u003c/b\u003e, 1759\u0026ndash;1788 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMemish ZA, \u003cem\u003eet al.\u003c/em\u003e Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg Infect Dis \u003cb\u003e19\u003c/b\u003e, 1819\u0026ndash;1823 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeroy EM, \u003cem\u003eet al.\u003c/em\u003e Fruit bats as reservoirs of Ebola virus. Nature \u003cb\u003e438\u003c/b\u003e, 575\u0026ndash;576 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunster VJ, \u003cem\u003eet al.\u003c/em\u003e Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis). Sci Rep \u003cb\u003e6\u003c/b\u003e, 21878 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou P, \u003cem\u003eet al.\u003c/em\u003e A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature \u003cb\u003e579\u003c/b\u003e, 270\u0026ndash;273 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMallapaty S. Closest known relatives of virus behind COVID-19 found in Laos. Nature \u003cb\u003e597\u003c/b\u003e, 603 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchountz T. Immunology of bats and their viruses: challenges and opportunities. Viruses \u003cb\u003e6\u003c/b\u003e, 4880\u0026ndash;4901 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe S, \u003cem\u003eet al.\u003c/em\u003e Bat coronaviruses and experimental infection of bats, the Philippines. Emerg Infect Dis \u003cb\u003e16\u003c/b\u003e, 1217\u0026ndash;1223 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubudhi S, \u003cem\u003eet al.\u003c/em\u003e A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat. J Gen Virol \u003cb\u003e98\u003c/b\u003e, 2297\u0026ndash;2309 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong S, \u003cem\u003eet al.\u003c/em\u003e New world bats harbor diverse influenza A viruses. PLoS Pathog \u003cb\u003e9\u003c/b\u003e, e1003657 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarding SR, Davis N, Hoyles L. Review article: the human intestinal virome in health and disease. Aliment Pharmacol Ther \u003cb\u003e46\u003c/b\u003e, 800\u0026ndash;815 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVemuri R, Shankar EM, Chieppa M, Eri R, Kavanagh K. Beyond Just Bacteria: Functional Biomes in the Gut Ecosystem Including Virome, Mycobiome, Archaeome and Helminths. Microorganisms \u003cb\u003e8\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu\u0026ntilde;oz-Fontela C, \u003cem\u003eet al.\u003c/em\u003e Animal models for COVID-19. Nature \u003cb\u003e586\u003c/b\u003e, 509\u0026ndash;515 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee A, Misra V, Schountz T, Baker ML. Tools to study pathogen-host interactions in bats. Virus Res \u003cb\u003e248\u003c/b\u003e, 5\u0026ndash;12 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClevers H. Modeling Development and Disease with Organoids. Cell \u003cb\u003e165\u003c/b\u003e, 1586\u0026ndash;1597 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxena K, \u003cem\u003eet al.\u003c/em\u003e Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology. J Virol \u003cb\u003e90\u003c/b\u003e, 43\u0026ndash;56 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Souza N. Organoids. Nature Methods \u003cb\u003e15\u003c/b\u003e, 23\u0026ndash;23 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRookmaaker MB, Schutgens F, Verhaar MC, Clevers H. Development and application of human adult stem or progenitor cell organoids. Nature Reviews Nephrology \u003cb\u003e11\u003c/b\u003e, 546 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlutt SE, Crawford SE, Ramani S, Zou WY, Estes MK. Engineered Human Gastrointestinal Cultures to Study the Microbiome and Infectious Diseases. Cell Mol Gastroenterol Hepatol \u003cb\u003e5\u003c/b\u003e, 241\u0026ndash;251 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell RH, Behnke MS. WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals. Biol Open \u003cb\u003e6\u003c/b\u003e, 698\u0026ndash;705 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbadawy M, \u003cem\u003eet al.\u003c/em\u003e Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus. Int J Mol Sci \u003cb\u003e22\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei X, \u003cem\u003eet al.\u003c/em\u003e Activation and evasion of type I interferon responses by SARS-CoV-2. Nature Communications \u003cb\u003e11\u003c/b\u003e, 3810 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, \u003cem\u003eet al.\u003c/em\u003e Infection of bat and human intestinal organoids by SARS-CoV-2. Nature Medicine \u003cb\u003e26\u003c/b\u003e, 1077\u0026ndash;1083 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClayton E, Munir M. Fundamental Characteristics of Bat Interferon Systems. Front Cell Infect Microbiol \u003cb\u003e10\u003c/b\u003e, 527921 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe La Cruz-Rivera PC, \u003cem\u003eet al.\u003c/em\u003e The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction. J Immunol \u003cb\u003e200\u003c/b\u003e, 209\u0026ndash;217 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubudhi S, Rapin N, Misra V. Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover. \u003cem\u003eViruses\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrving AT, \u003cem\u003eet al.\u003c/em\u003e Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection. Cell Rep \u003cb\u003e33\u003c/b\u003e, 108345 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkis S, \u003cem\u003eet al.\u003c/em\u003e A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR. Dev Comp Immunol \u003cb\u003e85\u003c/b\u003e, 95\u0026ndash;107 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie J, \u003cem\u003eet al.\u003c/em\u003e Dampened STING-Dependent Interferon Activation in Bats. Cell Host Microbe \u003cb\u003e23\u003c/b\u003e, 297\u0026ndash;301 e294 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranz KM, Neidermyer WJ, Tan YJ, Whelan SPJ, Kagan JC. STING-dependent translation inhibition restricts RNA virus replication. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cb\u003e115\u003c/b\u003e, E2058-E2067 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachain-Williams C, \u003cem\u003eet al.\u003c/em\u003e Serologic evidence of flavivirus infection in bats in the Yucatan Peninsula of Mexico. J Wildl Dis \u003cb\u003e49\u003c/b\u003e, 684\u0026ndash;689 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrumpf AA, Malmlov A, Ayers JD, Schountz T, Kendall LV. Hematologic Values of Jamaican Fruit Bats (Artibeus jamaicensis) and the Effects of Isoflurane Anesthesia. Journal of the American Association for Laboratory Animal Science: JAALAS \u003cb\u003e59\u003c/b\u003e, 275\u0026ndash;281 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerrard DL, \u003cem\u003eet al.\u003c/em\u003e Transcriptomic Signatures of Tacaribe Virus-Infected Jamaican Fruit Bats. \u003cem\u003emSphere\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalmlov A, \u003cem\u003eet al.\u003c/em\u003e Experimental Zika virus infection of Jamaican fruit bats (Artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells. PLoS Negl Trop Dis \u003cb\u003e13\u003c/b\u003e, e0007071 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReid JE, Jackson AC. Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants. J Neurovirol \u003cb\u003e7\u003c/b\u003e, 511\u0026ndash;517 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw TI, \u003cem\u003eet al.\u003c/em\u003e Transcriptome sequencing and annotation for the Jamaican fruit bat (Artibeus jamaicensis). PLoS One \u003cb\u003e7\u003c/b\u003e, e48472 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Tian S, Galindo-Gonzalez J, Davalos LM, Zhang Y, Zhao H. Molecular adaptation and convergent evolution of frugivory in Old World and neotropical fruit bats. Mol Ecol \u003cb\u003e29\u003c/b\u003e, 4366\u0026ndash;4381 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyoshi H, Stappenbeck TS. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nature protocols \u003cb\u003e8\u003c/b\u003e, 2471\u0026ndash;2482 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorten MA, Bajikar SS, Sasaki N, Clevers H, Janes KA. Automated brightfield morphometry of 3D organoid populations by OrganoSeg. Sci Rep \u003cb\u003e8\u003c/b\u003e, 5319 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKip AM, \u003cem\u003eet al.\u003c/em\u003e Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis \u003cb\u003e12\u003c/b\u003e, 95 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamers MM, \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 productively infects human gut enterocytes. Science \u003cb\u003e369\u003c/b\u003e, 50\u0026ndash;54 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTriana S, \u003cem\u003eet al.\u003c/em\u003e Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. Mol Syst Biol \u003cb\u003e17\u003c/b\u003e, e10232 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang KK, \u003cem\u003eet al.\u003c/em\u003e Variable susceptibility of intestinal organoid-derived monolayers to SARS-CoV-2 infection. PLoS Biol \u003cb\u003e20\u003c/b\u003e, e3001592 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfel R, \u003cem\u003eet al.\u003c/em\u003e Virological assessment of hospitalized patients with COVID-2019. Nature \u003cb\u003e581\u003c/b\u003e, 465\u0026ndash;469 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. \u003cb\u003e22\u003c/b\u003e, 2243\u0026ndash;2252 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanifer ML, \u003cem\u003eet al.\u003c/em\u003e Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells. Cell Rep \u003cb\u003e32\u003c/b\u003e, 107863 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanifer ML, Guo C, Doldan P, Boulant S. Importance of Type I and III Interferons at Respiratory and Intestinal Barrier Surfaces. Front Immunol \u003cb\u003e11\u003c/b\u003e, 608645 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou P, \u003cem\u003eet al.\u003c/em\u003e Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity. J Immunol \u003cb\u003e186\u003c/b\u003e, 3138\u0026ndash;3147 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSearle BC, \u003cem\u003eet al.\u003c/em\u003e Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun \u003cb\u003e9\u003c/b\u003e, 5128 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePruitt KD, \u003cem\u003eet al.\u003c/em\u003e RefSeq: an update on mammalian reference sequences. Nucleic Acids Res \u003cb\u003e42\u003c/b\u003e, D756-763 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhAinle M, \u003cem\u003eet al.\u003c/em\u003e A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV. Elife \u003cb\u003e7\u003c/b\u003e, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen EY, \u003cem\u003eet al.\u003c/em\u003e Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics \u003cb\u003e14\u003c/b\u003e, 128 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Zhang L, Li P, Zheng Y, Yang Y, Ji S. Apelin/APJ system in inflammation. Int Immunopharmacol \u003cb\u003e109\u003c/b\u003e, 108822 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouhaddou M, \u003cem\u003eet al.\u003c/em\u003e The Global Phosphorylation Landscape of SARS-CoV-2 Infection. Cell \u003cb\u003e182\u003c/b\u003e, 685\u0026ndash;712 e619 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiva L, \u003cem\u003eet al.\u003c/em\u003e Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing. Nature \u003cb\u003e586\u003c/b\u003e, 113\u0026ndash;119 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaggen J, \u003cem\u003eet al.\u003c/em\u003e Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2. Nat Genet \u003cb\u003e53\u003c/b\u003e, 435\u0026ndash;444 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamage AM, \u003cem\u003eet al.\u003c/em\u003e Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles. PLoS Pathog \u003cb\u003e16\u003c/b\u003e, e1009130 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeDiego ML, \u003cem\u003eet al.\u003c/em\u003e Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis. PLoS Pathog \u003cb\u003e7\u003c/b\u003e, e1002315 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlottau K, \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study. Lancet Microbe \u003cb\u003e1\u003c/b\u003e, e218-e225 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall JS, \u003cem\u003eet al.\u003c/em\u003e Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2. Transbound Emerg Dis \u003cb\u003e68\u003c/b\u003e, 3443\u0026ndash;3452 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin WW, Tsay AJ, Lalime EN, Pekosz A, Griffin DE. Primary differentiated respiratory epithelial cells respond to apical measles virus infection by shedding multinucleated giant cells. Proc Natl Acad Sci U S A \u003cb\u003e118\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAicher SM, \u003cem\u003eet al.\u003c/em\u003e Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells. J Virol \u003cb\u003e96\u003c/b\u003e, e0060822 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan H, \u003cem\u003eet al.\u003c/em\u003e ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species. Nat Ecol Evol \u003cb\u003e5\u003c/b\u003e, 600\u0026ndash;608 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson BA, \u003cem\u003eet al.\u003c/em\u003e Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. Nature \u003cb\u003e591\u003c/b\u003e, 293\u0026ndash;299 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchountz T, Baker ML, Butler J, Munster V. Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans. Front Immunol \u003cb\u003e8\u003c/b\u003e, 1098 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZang R, \u003cem\u003eet al.\u003c/em\u003e TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol \u003cb\u003e5\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrook CE, \u003cem\u003eet al.\u003c/em\u003e Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence. Elife \u003cb\u003e9\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosbauer K, \u003cem\u003eet al.\u003c/em\u003e The Effect of Allicin on the Proteome of SARS-CoV-2 Infected Calu-3 Cells. Front Microbiol \u003cb\u003e12\u003c/b\u003e, 746795 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVidova V, Spacil Z. A review on mass spectrometry-based quantitative proteomics: Targeted and data independent acquisition. Anal Chim Acta \u003cb\u003e964\u003c/b\u003e, 7\u0026ndash;23 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai YH, \u003cem\u003eet al.\u003c/em\u003e A Method for Cryogenic Preservation of Human Biopsy Specimens and Subsequent Organoid Culture. Cell Mol Gastroenterol Hepatol \u003cb\u003e6\u003c/b\u003e, 218\u0026ndash;222 e217 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebrell TA, \u003cem\u003eet al.\u003c/em\u003e Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events. Cell Tissue Res \u003cb\u003e371\u003c/b\u003e, 293\u0026ndash;307 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebrell TA, \u003cem\u003eet al.\u003c/em\u003e A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium. Cell Mol Gastroenterol Hepatol \u003cb\u003e8\u003c/b\u003e, 157\u0026ndash;171 e153 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHedges JF, \u003cem\u003eet al.\u003c/em\u003e An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model. Front Immunol \u003cb\u003e13\u003c/b\u003e, 918881 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoveday EK, \u003cem\u003eet al.\u003c/em\u003e Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure. Viruses \u003cb\u003e13\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRidley RG. \u003cem\u003eAntibodies: A Laboratory Manual\u003c/em\u003e, 2009/04/14 edn. Cambridge University Press (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGessulat S, \u003cem\u003eet al.\u003c/em\u003e Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning. Nat Methods \u003cb\u003e16\u003c/b\u003e, 509\u0026ndash;518 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Riverol Y, \u003cem\u003eet al.\u003c/em\u003e The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res \u003cb\u003e47\u003c/b\u003e, D442-D450 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraw S, \u003cem\u003eet al.\u003c/em\u003e proteiNorm - A User-Friendly Tool for Normalization and Analysis of TMT and Label-Free Protein Quantification. ACS Omega \u003cb\u003e5\u003c/b\u003e, 25625\u0026ndash;25633 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitchie ME, \u003cem\u003eet al.\u003c/em\u003e limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res \u003cb\u003e43\u003c/b\u003e, e47 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuleshov MV, \u003cem\u003eet al.\u003c/em\u003e The COVID-19 Drug and Gene Set Library. Patterns (N Y) \u003cb\u003e1\u003c/b\u003e, 100090 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2340919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2340919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBats 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.\u003c/p\u003e","manuscriptTitle":"Antiviral response mechanisms in a Jamaican Fruit Bat intestinal organoid model of SARS-CoV-2 infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-12 18:35:50","doi":"10.21203/rs.3.rs-2340919/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e622141-7d3b-488a-9b4f-d558ebaa25c9","owner":[],"postedDate":"December 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":17596239,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"},{"id":17596240,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2023-10-29T07:11:39+00:00","versionOfRecord":{"articleIdentity":"rs-2340919","link":"https://doi.org/10.1038/s41467-023-42610-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-10-28 04:00:00","publishedOnDateReadable":"October 28th, 2023"},"versionCreatedAt":"2022-12-12 18:35:50","video":"","vorDoi":"10.1038/s41467-023-42610-x","vorDoiUrl":"https://doi.org/10.1038/s41467-023-42610-x","workflowStages":[]},"version":"v1","identity":"rs-2340919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2340919","identity":"rs-2340919","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}