Equine coronavirus infection and replication in equine intestinal enteroids

Equine coronavirus infection and replication in equine intestinal enteroids | 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 Research Article Equine coronavirus infection and replication in equine intestinal enteroids Yoshinori Kambayashi, Manabu Nemoto, Akihiro Ochi, Daiki Kishi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4540308/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Oct, 2024 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract In this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as in vitro models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed in vivo in the intestinal epithelium. qRT-PCR and electron microscopy showed that ECoV had infected and replicated in the EIEs. These results suggest that EIEs can be novel in vitro tools for studying the interaction between equine intestinal epithelium and ECoV. (88 words) equine coronavirus equine intestinal enteroid horse viral infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction, Methods and Results Equine coronavirus (ECoV) primarily infects the small and large intestines of horses, resulting in fever, anorexia, lethargy, and diarrhea. [ 1 , 2 ]. Most affected horses exhibit only mild clinical signs, but in rare instances, ECoV causes fatal enteritis with severe watery diarrhea or neurological disorders due to hyperammonemia [ 3 , 4 ]. In most in vitro studies of ECoV infection, HRT-18G cells derived from human rectal adenocarcinoma have been used, because there is no cell line derived from equine intestinal epithelium [ 5 – 7 ]. Although HRT-18G cells support ECoV replication, they may not fully mimic the physiological properties of horses in vivo . Therefore, it is desirable to develop an in vitro system that can closely recapitulate the equine intestinal epithelium for further research on ECoV pathogenesis. Intestinal enteroids have recently been developed as novel in vitro models with which to investigate host–virus interactions in humas and animals [ 8 – 11 ]. They are derived from undifferentiated stem cells located in the crypt bases of the intestines with the capacity for self-renewal and multipotency [ 12 ]. They construct three-dimensional (3D) structures composed of various cell types expressed in vivo in the intestinal epithelium and recapitulate physiological activity of the intestinal epithelium in vivo [ 13 ]. Recently, equine intestinal enteroids (EIEs) have been established from equine jejunal tissues and used to evaluate immune responses [ 14 – 16 ]. However, to our knowledge, there has been no report describing the infectivity of enteric viruses in EIEs. Here, we generated EIEs from the small intestinal tissues of the duodenum, jejunum, and ileum, and investigated their suitability as in vitro models with which to study ECoV infection. Intestinal crypts were harvested from a 3-year-old Thoroughbred horse without gastrointestinal diseases that had been euthanized for reasons unrelated to this study. Crypts were isolated according to a protocol for mouse and human tissues, with some modifications [ 13 ]. In brief, approximately 3-cm each of the duodenum, jejunum, and ileum tissue resected immediately after euthanasia. They were chopped into 5-mm pieces and washed vigorously in ice-cold Dulbecco’s phosphate-buffered saline (D-PBS) (−) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) until the supernatant became clear. The fragments were incubated in ice-cold D-PBS (−) containing 5 mM EDTA on ice for 30 min and vigorously pipetted to detach the crypts from the epithelium. The supernatant was filtrated through a 100-µm cell strainer to remove villi. The filtrate was then centrifuged for 5 min at 400 × g at 4°C and the pelleted crypts were resuspended in 25 µL of ice-cold Matrigel (Corning, NY, USA). Matrigel containing the crypts was dropped onto a pre-warmed 48-well plate (Thermo Fisher Scientific, MA, USA). For tissue culture, Advanced DMEM/F-12 medium containing 10 mM HEPES, 1 × GlutaMAX (all from Thermo Fisher Scientific), and a mixed solution of 100 units/mL of penicillin and 100 µg/mL of streptomycin (Nacalai Tesque, Inc., Kyoto, Japan) was prepared as a basal medium. The following growth factors were added to the basal medium to create expansion medium for the maintenance of the stem cell niches and for cell proliferation: 1/10 Afamin/Wnt3a CM (MBL, Tokyo, Japan); 100 ng/mL Noggin (Peprotech, NJ, USA); 500 ng/mL R-spondin, 500 nM A83-01 (both from R&D Systems, MN, USA); 1 × B27, 1 × N2 supplement, 10 nM Gastrin Ⅰ, 50 ng/mL mouse recombinant EGF protein, 10 µM Y-27632,10 µM SB202190 (all from Thermo Fisher Scientific); 1 mM N-acetylcysteine (FUJIFILM Wako); and 2.5 µM CHIR (Cayman Chemical, MI, USA). Following the polymerization of the Matrigel for 15 min at 37°C, 250 µL of expansion medium was added and the plates were incubated at 37°C in 5% CO 2 . Expansion medium was refreshed every 3–4 days and EIEs were passaged at intervals of approximately 5–8 days. After the overlaid medium was removed, the Matrigel domes were mechanically disrupted with ice-cold basal medium. The resuspended EIEs were further dissociated and centrifuged for 5 min at 400 × g at 4°C. The pelleted EIEs were resuspended in Matrigel and cultured. To confirm that the EIEs contained the various cell types expressed in vivo in equine intestinal epithelium, we evaluated them by immunohistochemistry (IHC) and Alcian Blue Periodic Acid Schiff (AB-PAS) staining. EIEs were recovered from the Matrigel and solidified with iPgell (GenoStaff, Tokyo, Japan) according to the manufacturer’s instructions, fixed with formalin. After embedding in paraffin, 3-µm sections of the EIEs were stained with Histofine Simple Stain MAX PO Multi (Nichirei Biosciences, Tokyo, Japan). Primary antibodies were raised against villin (1:2, IR076, Agilent Technologies) for enterocytes, chromogranin A (CgA) (1:2000, 1–28, Yanaihara, Shizuoka, Japan) for enteroendocrine cells, Lysozyme (LYZ) (1:10, Nichirei Biosciences) for Paneth cells, Sox9 (1:4000, AB5535, Millipore, MA, USA) for stem cells, and Ki67 (1:500, ab15580, Abcam, Cambridge, UK) for proliferative cells. Following automated deparaffinization, heat-antigen retrieval was performed. The sections were incubated with the primary antibodies diluted with Antibody Diluent (Abcam) for 1 h at room temperature. Antibody binding was visualized by the addition of 3,3'-diaminobenzidine (Abcam) according to the manufacturer's protocol. Mucus secreted by goblet cells was stained with AB-PAS. Jejunal tissues of a healthy horse were also stained as positive controls, and tissues incubated without the primary antibody were used as negative controls. For virus inoculation to evaluate the susceptibility of EIEs to ECoV, the proliferated 3D EIEs were transferred to 2D monolayers on a 48-well flat-bottomed plate. The bottom of the plates had been pre-coated with 200 µL of 2.5% Matrigel diluted with D-PBS (−) at 37°C for 90 min. The EIEs recovered from the Matrigel were centrifuged and resuspended in 1 mL of TrypLE Express (Thermo Fisher Scientific). Following incubation at 37°C for 10 min, the cells were further pipetted for complete dissociation into single cells, and basal medium was added to halt the enzyme reaction. The suspension was centrifuged, and the cells were resuspended in expansion medium. After removal of the Matrigel solution, the dissociated cells were seeded at 1–2 × 10 4 cells/cm 2 and incubated at 37°C in 5% CO 2 . The expansion medium was refreshed the following day, and the monolayer culture was maintained for an additional 2–3 days until use. Monolayer EIEs that reached > 80% confluency were used in the virus inoculation test. ECoV strain NC99, isolated from a diarrheic Arabian foal [ 5 ], was used. The monolayer EIEs were washed once with 100 µL of basal medium and inoculated with ECoV at multiplicity of infection (MOI) of 0.1. After incubation for 60 min at 37°C in 5% CO 2 , the cells were washed twice with 100 µL of basal medium, and 250 µL of expansion medium was added. The overlaid medium samples were collected from three wells at 1, 6, 12, 24, 48, and 72 h post-inoculation (hpi) and subjected to quantitative real-time reverse-transcription PCR (qRT-PCR) to assess viral replication. For evaluation by electron microscopy, dissociated jejunal EIEs seeded at 2 × 10 4 cells/well in a Matrigel-precoated 24-well plate were inoculated with NC99 at MOI of 0.1. ECoV RNA in the collected culture medium was quantified by qRT-PCR. Total RNA was extracted in magLEAD 12gc automated nucleic acid extraction system (Precision System Science, Japan). qRT-PCR was performed on a StepOnePlus qPCR system (Applied Biosystems, CA, USA), using TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific). We used specific primer sets (ECoV-380f 5’-TGG GAA CAG GCC CGC-3’ and ECoV-522r 5’-CCT AGT CGG AAT AGC CTC ATCAC-3’) and TaqMan MGB probe (ECoV-436p 5’-6-FAM-TGG GTC GCT AAC AAG-MGB-3’) (Thermo Fisher Scientific) to detect the nucleocapsid ( N ) gene of ECoV [ 17 ]. The thermal cycling conditions were an initial hold at 25°C for 2 min, 50°C for 15 min and 95°C for 2 min, and then 40 cycles of 95°C for 3 s and 60°C for 30 s. All tests were performed in triplicate for each sample, and the average copy numbers were determined. For electron microscopy, the jejunal EIEs inoculated with ECoV were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at 72 hpi. They were then postfixed with 2% osmium tetroxide in 0.1 M PB at 4°C for 1 h, and embedded in Quetol-812 (Nisshin EM Co, Japan). Ultrathin sections were stained with 2% uranyl acetate and Lead stain solution (Sigma-Aldrich Co., MO, USA) and examined with a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Japan). The post fixation and subsequent processes were performed by Tokai Electron Microscopy, Inc. (Aichi, Japan). Crypts isolated from the duodenum, jejunum, and ileum started to form bud structures after 6–7 days of the culture (Fig. 1 A). After further development over another 2–3 days, they formed more complex and multilobular structures. The passaged EIEs formed multilobular structures and reached a significant size by days 5–8 (Fig. 1 B). The optimal split ratio was between 1:6 and 1:10, varying with the number and size of the EIEs. The EIEs could be passaged at least 15 times without visible morphological changes or a decline in proliferation (Fig. 1 C). IHC showed that a substantial number of Sox9-positive cells were observed throughout the EIEs, as well as Ki-67 positive proliferative cells (Fig. 2 ). Villin-positive enterocytes were observed throughout the luminal surface of the EIEs. In addition, CgA-positive enteroendocrine cells, LYZ-positive Paneth cells, and AB-PAS-positive Goblet cells were identified. These cell types were also identified in the tissue sections of healthy jejunum, but no positive cells were observed in negative control samples (data not shown). The copy number of ECoV RNA had started to increase at 6 hpi and continued to increase up to 72 hpi in monolayer EIEs (Fig. 3 ). At 72 hpi, the copy numbers had increased relative to that of 2 hpi by 3563× in the duodenal EIEs, by 8043× in the jejunal EIEs, and by 3201× in the ileal EIEs. No cytopathic effect was observed in any of the EIEs. Viral proliferation was also evaluated by electron microscopy in monolayer EIEs derived from the jejunum. Virus particles were observed within the cytoplasm or cytoplasmic vesicles (Fig. 4 A, B). Moreover, numerous virus particles were identified on the cell membrane, and they were budding from the surface of the cell membrane and releasing into the extracellular space (Fig. 4 C–E). Discussion We cultured equine intestinal crypts of the small intestine (duodenum, jejunum, and ileum) following the method used with human and mouse tissues [ 13 ]. The crypts formed 3D cell structures and could be passaged at least 15 times without visible morphological changes. IHC confirmed that the generated EIEs were composed of a large number of undifferentiated stem cells. This result is reasonable given that the culture medium used in this study contained growth factors intended to maintain the stem cell niche, including Wnt3a, R-spondin, and Noggin. In addition, differentiated enterocytes, enteroendocrine cells, goblet cells, and Paneth cells were identified. Villin, a marker protein of the brush border of enterocytes, was expressed on the luminal surface of the EIEs, indicating that the cellular polarity was kept in their 3D structures. Thus, IHC identified all cell types expressed in vivo in the small intestinal epithelium in the EIEs, demonstrating that EIEs could be generated from a variety of equine small intestinal tissues. However, the EIEs in our study may have not completely recapitulated the cellular composition of the small intestinal epithelium in vivo , because stem cells originally located in the intestinal crypt bases were most representative (Fig. 2 ). The differentiation of intestinal enteroids can be accelerated by reducing or removing Wnt, R-spondin, and Noggin in human and murine enteroids [ 12 , 13 ]. Although we tried this, the proliferation rate was declined (data not shown), so we did not use the technique in this study. Most recently, appropriate culture conditions that promote the differentiation of enteroids from the equine jejunum and colon have been reported [ 18 ]. The differentiation of EIEs should make it possible to recapitulate in vivo cellular composition more accurately and advance further research of host–virus interactions. Intestinal enteroids that form 3D structures following cellular polarity face their apical surface to the inside of the structure, making it challenging to expose their luminal side to experimental agents. In several studies, intestinal enteroids were mechanically sheared to expose the luminal surface and inoculated with viral pathogens [ 10 , 19 ]. However, there is a concern that this method does not accurately recapitulate the in vivo situation, as the basal side of cells is also exposed. By contrast, a 2D monolayer model in which enteroid-derived cells are seeded on flat-bottomed plates allows only the apical surface to be exposed to experimental agents. This approach is therefore commonly preferred in studies of host–virus interactions in humans and porcine [ 9 , 20 , 21 ]. Here, we used this approach to inoculate EIEs with ECoV. EIEs derived from any tissue (duodenum, jejunum, or ileum) could be transferred into a 2D monolayer. The cell density reached > 80% after 3–4 days in monolayer culture and the cell confluency was maintained during the study period. Therefore, a cell concentration of 1–2 × 10 4 cells/cm 2 should be the most appropriate for monolayer culture in our protocol. The large increase of ECoV RNA in all EIEs shows that ECoV robustly replicated in the EIEs. In addition, electron microscopy revealed the presence of virus-like particles in the cytoplasm, and a considerable number of viruses were budding from the cell membrane of the jejunal EIEs. These findings indicate that the viruses replicated inside the cell and were released from the cell membrane into the extracellular space.Thus, ECoV can infect EIEs derived from all three regions of the small intestine and substantially replicate. To our knowledge, this is the first report describing enteric virus infection in EIEs. Our previous experimental challenge study in horses showed that ECoV infected throughout the small and large intestinal tissues, as confirmed by RT-qPCR and in situ hybridization [ 1 ]. The current study reproduced the in vivo situation, showing that EIEs offer a promise as a novel in vitro model for studying ECoV infection. In humans and other animals, the potential applications of intestinal enteroids include the identification of the target cells of enteric pathogens, the elucidation of infectious mechanisms, and the isolation of viruses from clinical specimen [ 8 , 9 , 22 ]. To advance these studies in EIEs, further research is needed. One limitation of the current study is that EIEs were generated from the tissues of only one horse. It is important to note that the intestinal enteroids originate from the donor’s tissues and therefore may exhibit characteristics inherited from those tissues. It is probable that enteroids derived from different individuals differ in susceptibility to enteric pathogens. For example, the growth rate of human rotavirus differed among intestinal enteroids derived from individual humans [ 22 ]. Therefore, it is preferable to generate EIEs from multiple individuals and compare their capacity to sustain viral infection in order to confirm whether our results are representative of EIEs in general. In conclusion, we generated duodenal, jejunal, and ileal enteroids from horse tissues and demonstrated that ECoV infects and replicates in these enteroids in a 2D monolayer model. EIEs offer promise as a robust tool with which to investigate interactions between the equine intestinal epithelium and ECoV in vitro . Declarations Ethics approval and consent to participate The experimental protocol and all animal procedures were approved by the Animal Care Committee of the Equine Research Institute of the Japan Racing Association with approved numbers 21-6. Consent for publication Not applicable. Availability of data and materials The datasets used and analyzed in the study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding Our research was funded by the Japan Racing Association. Authors’ contributions YK participated in all experiments and drafted the manuscript. MN and TS contributed to design and supervision of this study. AO, DK, and TU performed all histological analyses. KT supported RT-qPCR analysis. HB, MO, and NK reviewed the manuscript and provided advice. Acknowledgements NC99 was provided by James S. Guy, a professor of veterinary virology at North Carolina University, in 2001. We are grateful to Akira Kokubun, Akiko Kasagawa, Miwa Tanaka, and Kaoru Watanabe (Equine Research Institute, Japan Racing Association) for their invaluable technical assistance. References Kambayashi Y, Kishi D, Ueno T, Ohta M, Bannai H, Tsujimura K, Kinoshita Y, Nemoto M (2022) Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses. Arch Virol 167:1611–1618 Kambayashi Y, Bannai H, Tsujimura K, Hirama A, Ohta M, Nemoto M (2021) Outbreak of equine coronavirus infection among riding horses in Tokyo, Japan, Comp Immunol Microbiol Infect Dis. 77:101668 Fielding CL, Higgins JK, Higgins JC, McIntosh S, Scott E, Giannitti F, Mete A, Pusterla N (2015) Disease associated with equine coronavirus infection and high case fatality rate. J Vet Intern Med 29:307–310 Giannitti F, Diab S, Mete A, Stanton JB, Fielding L, Crossley B, Sverlow K, Fish S, Mapes S, Scott L, Pusterla N (2015) Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids, Vet Pathol. 52:1148–1156 Guy JS, Breslin JJ, Breuhaus B, Vivrette S, Smith LG (2000) Characterization of a coronavirus isolated from a diarrheic foal. J Clin Microbiol 38:4523–4526 Oue Y, Morita Y, Kondo T, Nemoto M (2013) Epidemic of equine coronavirus at Obihiro Racecourse, Hokkaido, Japan in 2012. J Vet Med Sci 75:1261–1265 Oue Y, Ishihara R, Edamatsu H, Morita Y, Yoshida M, Yoshima M, Hatama S, Murakami K, Kanno T (2011) Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain. Vet Microbiol 150:41–48 Ettayebi K, Crawford SE, Murakami K, Broughman JR, Karandikar U, Tenge VR, Neill FH, Blutt SE, Zeng XL, Qu L, Kou B, Opekun AR, Burrin D, Graham DY, Ramani S, Atmar RL, Estes MK (2016) Replication of human noroviruses in stem cell-derived human enteroids. Science 353:1387–1393 Li L, Fu F, Guo S, Wang H, He X, Xue M, Yin L, Feng L, Liu P (2019) Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response. J Virol. 93 Zhou J, Li C, Liu X, Chiu MC, Zhao X, Wang D, Wei Y, Lee A, Zhang AJ, Chu H, Cai JP, Yip CC, Chan IH, Wong KK, Tsang OT, Chan KH, Chan JF, To KK, Chen H, Yuen KY (2020) Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med 26:1077–1083 Tekes G, Ehmann R, Boulant S, Stanifer ML (2020) Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection, Cells. 9 Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265 Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141:1762–1772 Stewart AS, Freund JM, Gonzalez LM (2018) Advanced three-dimensional culture of equine intestinal epithelial stem cells. Equine Vet J 50:241–248 Hellman S (2021) Generation of equine enteroids and enteroid-derived 2D monolayers that are responsive to microbial mimics. Vet Res 52:108 Hellman S, Martin F, Tydén E, Sellin ME, Norman A, Hjertner B, Svedberg P, Fossum C (2024) Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection. Vet Res 55:25 Pusterla N, Mapes S, Wademan C, White A, Ball R, Sapp K, Burns P, Ormond C, Butterworth K, Bartol J, Magdesian KG (2013) Emerging outbreaks associated with equine coronavirus in adult horses. Vet Microbiol 162:228–231 Windhaber C, Heckl A, Csukovich G, Pratscher B, Burgener IA, Biermann N, Dengler F (2024) A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Vet Res 55:30 Zhao X, Li C, Liu X, Chiu MC, Wang D, Wei Y, Chu H, Cai JP, Hau-Yee Chan I, Kak-Yuen Wong K, Fuk-Woo Chan J, Kai-Wang To K, Yuen KY, Zhou J (2021) Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus. Stem Cell Rep 16:493–504 Roodsant T, Navis M, Aknouch I, Renes IB, van Elburg RM, Pajkrt D, Wolthers KC, Schultsz C, van der Ark KCH, Sridhar A, Muncan V (2020) A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine. Front Cell Infect Microbiol 10:272 van der Hee B, Loonen LMP, Taverne N, Taverne-Thiele JJ, Smidt H, Wells JM (2018) Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids. Stem Cell Res 28:165–171 Saxena K, Blutt SE, Ettayebi K, Zeng XL, Broughman JR, Crawford SE, Karandikar UC, Sastri NP, Conner ME, Opekun AR, Graham DY, Qureshi W, Sherman V, Foulke-Abel J (2016) In J., Kovbasnjuk O., Zachos N.C., Donowitz M., Estes M.K., Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology, J Virol. 90:43–56 Cite Share Download PDF Status: Published Journal Publication published 10 Oct, 2024 Read the published version in Veterinary Research → 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4540308","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314629198,"identity":"0a4a28a8-fa8c-4b8a-bbc1-f3d13872647c","order_by":0,"name":"Yoshinori Kambayashi","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Yoshinori","middleName":"","lastName":"Kambayashi","suffix":""},{"id":314629199,"identity":"4d13ac7d-6e35-4e0a-a53c-7232c3af503f","order_by":1,"name":"Manabu Nemoto","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Manabu","middleName":"","lastName":"Nemoto","suffix":""},{"id":314629200,"identity":"a784daf5-dad2-4357-934f-d72bc2bb3ec8","order_by":2,"name":"Akihiro Ochi","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Ochi","suffix":""},{"id":314629201,"identity":"d57d5817-8bf3-4b1a-adf5-609e4d0efa5e","order_by":3,"name":"Daiki Kishi","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Kishi","suffix":""},{"id":314629202,"identity":"f322a668-6081-4bfc-8972-10ce6421818d","order_by":4,"name":"Takanori Ueno","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Takanori","middleName":"","lastName":"Ueno","suffix":""},{"id":314629203,"identity":"7082aa54-abd6-4d9e-80f6-962667d38a97","order_by":5,"name":"Koji Tsujimura","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Tsujimura","suffix":""},{"id":314629204,"identity":"2bd775d6-9f8a-495d-ade8-54f57707eb5a","order_by":6,"name":"Hiroshi Bannai","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Bannai","suffix":""},{"id":314629205,"identity":"92ba8503-3c29-4926-86df-3c58c1af3531","order_by":7,"name":"Nanako Kawanishi","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Nanako","middleName":"","lastName":"Kawanishi","suffix":""},{"id":314629206,"identity":"5a775052-9454-4d96-b95b-5e235a129a6f","order_by":8,"name":"Minoru Ohta","email":"","orcid":"","institution":"Equine Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Minoru","middleName":"","lastName":"Ohta","suffix":""},{"id":314629207,"identity":"168f0a8a-5deb-41a8-a242-6b165f14fcfe","order_by":9,"name":"Tohru Suzuki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYJACxgYDCR5+9gaGDxVQEQMitFjISfYcYJxxhngtDBXGBjMSEFrwAvkZ6c8kZxRIJG6QfPyw4eAeOwZ59waG4gI8WgxuJKRJbjCQSNwunWbYcOBZMoPhmQMMxjPwaZFIOCb5AKhl5+wE88cfDhxgMJyRwGDMg9dhiW1gLRtuHv/YcIAYLQw3ktlADjM2uMFjCNYiL0FAi8GZZ8yWMwwkgIGcUwjUksxjwHOwAa9f5NvTH97s+VMHjMrjG4Fa7OTk25uPGeMLMQzAY3CAsc2YFB1AexsYmB+TpmUUjIJRMAqGOQAApzNSS3Qf0eMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3607-4787","institution":"National Institute of Animal Health","correspondingAuthor":true,"prefix":"","firstName":"Tohru","middleName":"","lastName":"Suzuki","suffix":""}],"badges":[],"createdAt":"2024-06-06 12:22:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4540308/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4540308/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-024-01381-z","type":"published","date":"2024-10-10T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59599120,"identity":"410d5023-6342-4035-a5ad-59aa6f002021","added_by":"auto","created_at":"2024-07-03 16:30:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":369223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopy images of EIEs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(legend) \u003cstrong\u003eA\u003c/strong\u003eEnteroids at 10 days’ culture after isolation of crypts. \u003cstrong\u003eB\u003c/strong\u003e Representative images of the time course of ileal enteroids growth. \u003cstrong\u003eC\u003c/strong\u003e Duodenal and jejunal enteroids that were passaged 15 times and ileal enteroids that were passaged 17 times. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4540308/v1/455b4a99b3aed2753e3fb7f1.png"},{"id":59600123,"identity":"647ff595-184b-44ec-a9fa-f9876efecd21","added_by":"auto","created_at":"2024-07-03 16:38:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2273744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the cell types in EIEs and equine jejunal tissue by immunohistochemistry and AB-PAS staining.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(legend) EIEs derived from duodenal, jejunal, and ileal tissues were immunostained (shown as brown) with Sox9 as a cell marker of intestinal stem cells, Ki-67 for proliferative cells, villin for enterocytes, chromogranin A (CgA) for enteroendocrine cells, and lysozyme (LYZ) for Paneth cells. Mucin was stained by AB-PAS as a marker of goblet cells (magenta). Black arrow heads indicate positive stained cells. Sox9-positive cells were identified throughout the EIEs. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4540308/v1/e946aa779901ece9e9428b12.png"},{"id":59599118,"identity":"ef08b27b-0b3b-448b-9b97-eb14ea06cbfc","added_by":"auto","created_at":"2024-07-03 16:30:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinetics curve of ECoV replication in EIEs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(legend) Culture medium collected at 1, 6, 12, 24, 48, and 72 h after inoculation was subjected to RT-qPCR to determine viral gene copy number. The number of viral genes in 1 mL of original sample was calculated and expressed as the common logarithm. Data are presented as the means ± SD.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4540308/v1/20531210b89f99c63e29851e.png"},{"id":59600124,"identity":"c4648edd-f839-42f6-90c3-47766387ffc0","added_by":"auto","created_at":"2024-07-03 16:38:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":207381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectron microscopy images of the jejunal EIEs inoculated with ECoV.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(legend) Jejunal EIEs inoculated with ECoV were fixed and evaluated by electron microscopy. Virus particles were observed in the \u003cstrong\u003e(A) \u003c/strong\u003ecytoplasmic vesicles and \u003cstrong\u003e(B) \u003c/strong\u003ecytoplasm, and \u003cstrong\u003e(C-E) \u003c/strong\u003eto be budding from the surface of the cell membrane and releasing into the extracellular space. \u003cstrong\u003eD\u003c/strong\u003e is a magnified image of \u003cstrong\u003eC\u003c/strong\u003e. \u003cstrong\u003eE\u003c/strong\u003eis a magnified image of \u003cstrong\u003eD\u003c/strong\u003e. Scale is shown in each image.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4540308/v1/dd9f65a6818110714051df50.png"},{"id":66597342,"identity":"24762412-6fe2-4b6f-bd72-838d61eab907","added_by":"auto","created_at":"2024-10-14 16:09:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3269809,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4540308/v1/e6cf1bf7-982d-45ae-ab8e-e0c1ed1693cf.pdf"}],"financialInterests":"","formattedTitle":"Equine coronavirus infection and replication in equine intestinal enteroids","fulltext":[{"header":"Introduction, Methods and Results","content":"\u003cp\u003eEquine coronavirus (ECoV) primarily infects the small and large intestines of horses, resulting in fever, anorexia, lethargy, and diarrhea. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most affected horses exhibit only mild clinical signs, but in rare instances, ECoV causes fatal enteritis with severe watery diarrhea or neurological disorders due to hyperammonemia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In most \u003cem\u003ein vitro\u003c/em\u003e studies of ECoV infection, HRT-18G cells derived from human rectal adenocarcinoma have been used, because there is no cell line derived from equine intestinal epithelium [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although HRT-18G cells support ECoV replication, they may not fully mimic the physiological properties of horses \u003cem\u003ein vivo\u003c/em\u003e. Therefore, it is desirable to develop an \u003cem\u003ein vitro\u003c/em\u003e system that can closely recapitulate the equine intestinal epithelium for further research on ECoV pathogenesis. Intestinal enteroids have recently been developed as novel \u003cem\u003ein vitro\u003c/em\u003e models with which to investigate host\u0026ndash;virus interactions in humas and animals [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They are derived from undifferentiated stem cells located in the crypt bases of the intestines with the capacity for self-renewal and multipotency [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. They construct three-dimensional (3D) structures composed of various cell types expressed \u003cem\u003ein vivo\u003c/em\u003e in the intestinal epithelium and recapitulate physiological activity of the intestinal epithelium \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recently, equine intestinal enteroids (EIEs) have been established from equine jejunal tissues and used to evaluate immune responses [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, to our knowledge, there has been no report describing the infectivity of enteric viruses in EIEs. Here, we generated EIEs from the small intestinal tissues of the duodenum, jejunum, and ileum, and investigated their suitability as \u003cem\u003ein vitro\u003c/em\u003e models with which to study ECoV infection.\u003c/p\u003e \u003cp\u003eIntestinal crypts were harvested from a 3-year-old Thoroughbred horse without gastrointestinal diseases that had been euthanized for reasons unrelated to this study. Crypts were isolated according to a protocol for mouse and human tissues, with some modifications [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In brief, approximately 3-cm each of the duodenum, jejunum, and ileum tissue resected immediately after euthanasia. They were chopped into 5-mm pieces and washed vigorously in ice-cold Dulbecco\u0026rsquo;s phosphate-buffered saline (D-PBS) (\u0026minus;) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) until the supernatant became clear. The fragments were incubated in ice-cold D-PBS (\u0026minus;) containing 5 mM EDTA on ice for 30 min and vigorously pipetted to detach the crypts from the epithelium. The supernatant was filtrated through a 100-\u0026micro;m cell strainer to remove villi. The filtrate was then centrifuged for 5 min at 400 \u0026times; g at 4\u0026deg;C and the pelleted crypts were resuspended in 25 \u0026micro;L of ice-cold Matrigel (Corning, NY, USA). Matrigel containing the crypts was dropped onto a pre-warmed 48-well plate (Thermo Fisher Scientific, MA, USA). For tissue culture, Advanced DMEM/F-12 medium containing 10 mM HEPES, 1 \u0026times; GlutaMAX (all from Thermo Fisher Scientific), and a mixed solution of 100 units/mL of penicillin and 100 \u0026micro;g/mL of streptomycin (Nacalai Tesque, Inc., Kyoto, Japan) was prepared as a basal medium. The following growth factors were added to the basal medium to create expansion medium for the maintenance of the stem cell niches and for cell proliferation: 1/10 Afamin/Wnt3a CM (MBL, Tokyo, Japan); 100 ng/mL Noggin (Peprotech, NJ, USA); 500 ng/mL R-spondin, 500 nM A83-01 (both from R\u0026amp;D Systems, MN, USA); 1 \u0026times; B27, 1 \u0026times; N2 supplement, 10 nM Gastrin Ⅰ, 50 ng/mL mouse recombinant EGF protein, 10 \u0026micro;M Y-27632,10 \u0026micro;M SB202190 (all from Thermo Fisher Scientific); 1 mM N-acetylcysteine (FUJIFILM Wako); and 2.5 \u0026micro;M CHIR (Cayman Chemical, MI, USA). Following the polymerization of the Matrigel for 15 min at 37\u0026deg;C, 250 \u0026micro;L of expansion medium was added and the plates were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Expansion medium was refreshed every 3\u0026ndash;4 days and EIEs were passaged at intervals of approximately 5\u0026ndash;8 days. After the overlaid medium was removed, the Matrigel domes were mechanically disrupted with ice-cold basal medium. The resuspended EIEs were further dissociated and centrifuged for 5 min at 400 \u0026times; g at 4\u0026deg;C. The pelleted EIEs were resuspended in Matrigel and cultured.\u003c/p\u003e \u003cp\u003eTo confirm that the EIEs contained the various cell types expressed \u003cem\u003ein vivo\u003c/em\u003e in equine intestinal epithelium, we evaluated them by immunohistochemistry (IHC) and Alcian Blue Periodic Acid Schiff (AB-PAS) staining. EIEs were recovered from the Matrigel and solidified with iPgell (GenoStaff, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions, fixed with formalin. After embedding in paraffin, 3-\u0026micro;m sections of the EIEs were stained with Histofine Simple Stain MAX PO Multi (Nichirei Biosciences, Tokyo, Japan). Primary antibodies were raised against villin (1:2, IR076, Agilent Technologies) for enterocytes, chromogranin A (CgA) (1:2000, 1\u0026ndash;28, Yanaihara, Shizuoka, Japan) for enteroendocrine cells, Lysozyme (LYZ) (1:10, Nichirei Biosciences) for Paneth cells, Sox9 (1:4000, AB5535, Millipore, MA, USA) for stem cells, and Ki67 (1:500, ab15580, Abcam, Cambridge, UK) for proliferative cells. Following automated deparaffinization, heat-antigen retrieval was performed. The sections were incubated with the primary antibodies diluted with Antibody Diluent (Abcam) for 1 h at room temperature. Antibody binding was visualized by the addition of 3,3'-diaminobenzidine (Abcam) according to the manufacturer's protocol. Mucus secreted by goblet cells was stained with AB-PAS. Jejunal tissues of a healthy horse were also stained as positive controls, and tissues incubated without the primary antibody were used as negative controls.\u003c/p\u003e \u003cp\u003eFor virus inoculation to evaluate the susceptibility of EIEs to ECoV, the proliferated 3D EIEs were transferred to 2D monolayers on a 48-well flat-bottomed plate. The bottom of the plates had been pre-coated with 200 \u0026micro;L of 2.5% Matrigel diluted with D-PBS (\u0026minus;) at 37\u0026deg;C for 90 min. The EIEs recovered from the Matrigel were centrifuged and resuspended in 1 mL of TrypLE Express (Thermo Fisher Scientific). Following incubation at 37\u0026deg;C for 10 min, the cells were further pipetted for complete dissociation into single cells, and basal medium was added to halt the enzyme reaction. The suspension was centrifuged, and the cells were resuspended in expansion medium. After removal of the Matrigel solution, the dissociated cells were seeded at 1\u0026ndash;2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e and incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. The expansion medium was refreshed the following day, and the monolayer culture was maintained for an additional 2\u0026ndash;3 days until use.\u003c/p\u003e \u003cp\u003eMonolayer EIEs that reached\u0026thinsp;\u0026gt;\u0026thinsp;80% confluency were used in the virus inoculation test. ECoV strain NC99, isolated from a diarrheic Arabian foal [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], was used. The monolayer EIEs were washed once with 100 \u0026micro;L of basal medium and inoculated with ECoV at multiplicity of infection (MOI) of 0.1. After incubation for 60 min at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e, the cells were washed twice with 100 \u0026micro;L of basal medium, and 250 \u0026micro;L of expansion medium was added. The overlaid medium samples were collected from three wells at 1, 6, 12, 24, 48, and 72 h post-inoculation (hpi) and subjected to quantitative real-time reverse-transcription PCR (qRT-PCR) to assess viral replication. For evaluation by electron microscopy, dissociated jejunal EIEs seeded at 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in a Matrigel-precoated 24-well plate were inoculated with NC99 at MOI of 0.1.\u003c/p\u003e \u003cp\u003eECoV RNA in the collected culture medium was quantified by qRT-PCR. Total RNA was extracted in magLEAD 12gc automated nucleic acid extraction system (Precision System Science, Japan). qRT-PCR was performed on a StepOnePlus qPCR system (Applied Biosystems, CA, USA), using TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific). We used specific primer sets (ECoV-380f 5\u0026rsquo;-TGG GAA CAG GCC CGC-3\u0026rsquo; and ECoV-522r 5\u0026rsquo;-CCT AGT CGG AAT AGC CTC ATCAC-3\u0026rsquo;) and TaqMan MGB probe (ECoV-436p 5\u0026rsquo;-6-FAM-TGG GTC GCT AAC AAG-MGB-3\u0026rsquo;) (Thermo Fisher Scientific) to detect the nucleocapsid (\u003cem\u003eN\u003c/em\u003e) gene of ECoV [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The thermal cycling conditions were an initial hold at 25\u0026deg;C for 2 min, 50\u0026deg;C for 15 min and 95\u0026deg;C for 2 min, and then 40 cycles of 95\u0026deg;C for 3 s and 60\u0026deg;C for 30 s. All tests were performed in triplicate for each sample, and the average copy numbers were determined.\u003c/p\u003e \u003cp\u003eFor electron microscopy, the jejunal EIEs inoculated with ECoV were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at 72 hpi. They were then postfixed with 2% osmium tetroxide in 0.1 M PB at 4\u0026deg;C for 1 h, and embedded in Quetol-812 (Nisshin EM Co, Japan). Ultrathin sections were stained with 2% uranyl acetate and Lead stain solution (Sigma-Aldrich Co., MO, USA) and examined with a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Japan). The post fixation and subsequent processes were performed by Tokai Electron Microscopy, Inc. (Aichi, Japan).\u003c/p\u003e \u003cp\u003eCrypts isolated from the duodenum, jejunum, and ileum started to form bud structures after 6\u0026ndash;7 days of the culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After further development over another 2\u0026ndash;3 days, they formed more complex and multilobular structures. The passaged EIEs formed multilobular structures and reached a significant size by days 5\u0026ndash;8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The optimal split ratio was between 1:6 and 1:10, varying with the number and size of the EIEs. The EIEs could be passaged at least 15 times without visible morphological changes or a decline in proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). IHC showed that a substantial number of Sox9-positive cells were observed throughout the EIEs, as well as Ki-67 positive proliferative cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Villin-positive enterocytes were observed throughout the luminal surface of the EIEs. In addition, CgA-positive enteroendocrine cells, LYZ-positive Paneth cells, and AB-PAS-positive Goblet cells were identified. These cell types were also identified in the tissue sections of healthy jejunum, but no positive cells were observed in negative control samples (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe copy number of ECoV RNA had started to increase at 6 hpi and continued to increase up to 72 hpi in monolayer EIEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At 72 hpi, the copy numbers had increased relative to that of 2 hpi by 3563\u0026times; in the duodenal EIEs, by 8043\u0026times; in the jejunal EIEs, and by 3201\u0026times; in the ileal EIEs. No cytopathic effect was observed in any of the EIEs. Viral proliferation was also evaluated by electron microscopy in monolayer EIEs derived from the jejunum. Virus particles were observed within the cytoplasm or cytoplasmic vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Moreover, numerous virus particles were identified on the cell membrane, and they were budding from the surface of the cell membrane and releasing into the extracellular space (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe cultured equine intestinal crypts of the small intestine (duodenum, jejunum, and ileum) following the method used with human and mouse tissues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The crypts formed 3D cell structures and could be passaged at least 15 times without visible morphological changes. IHC confirmed that the generated EIEs were composed of a large number of undifferentiated stem cells. This result is reasonable given that the culture medium used in this study contained growth factors intended to maintain the stem cell niche, including Wnt3a, R-spondin, and Noggin. In addition, differentiated enterocytes, enteroendocrine cells, goblet cells, and Paneth cells were identified. Villin, a marker protein of the brush border of enterocytes, was expressed on the luminal surface of the EIEs, indicating that the cellular polarity was kept in their 3D structures. Thus, IHC identified all cell types expressed \u003cem\u003ein vivo\u003c/em\u003e in the small intestinal epithelium in the EIEs, demonstrating that EIEs could be generated from a variety of equine small intestinal tissues. However, the EIEs in our study may have not completely recapitulated the cellular composition of the small intestinal epithelium \u003cem\u003ein vivo\u003c/em\u003e, because stem cells originally located in the intestinal crypt bases were most representative (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The differentiation of intestinal enteroids can be accelerated by reducing or removing Wnt, R-spondin, and Noggin in human and murine enteroids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although we tried this, the proliferation rate was declined (data not shown), so we did not use the technique in this study. Most recently, appropriate culture conditions that promote the differentiation of enteroids from the equine jejunum and colon have been reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The differentiation of EIEs should make it possible to recapitulate \u003cem\u003ein vivo\u003c/em\u003e cellular composition more accurately and advance further research of host\u0026ndash;virus interactions.\u003c/p\u003e \u003cp\u003eIntestinal enteroids that form 3D structures following cellular polarity face their apical surface to the inside of the structure, making it challenging to expose their luminal side to experimental agents. In several studies, intestinal enteroids were mechanically sheared to expose the luminal surface and inoculated with viral pathogens [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, there is a concern that this method does not accurately recapitulate the \u003cem\u003ein vivo\u003c/em\u003e situation, as the basal side of cells is also exposed. By contrast, a 2D monolayer model in which enteroid-derived cells are seeded on flat-bottomed plates allows only the apical surface to be exposed to experimental agents. This approach is therefore commonly preferred in studies of host\u0026ndash;virus interactions in humans and porcine [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Here, we used this approach to inoculate EIEs with ECoV. EIEs derived from any tissue (duodenum, jejunum, or ileum) could be transferred into a 2D monolayer. The cell density reached\u0026thinsp;\u0026gt;\u0026thinsp;80% after 3\u0026ndash;4 days in monolayer culture and the cell confluency was maintained during the study period. Therefore, a cell concentration of 1\u0026ndash;2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e should be the most appropriate for monolayer culture in our protocol.\u003c/p\u003e \u003cp\u003eThe large increase of ECoV RNA in all EIEs shows that ECoV robustly replicated in the EIEs. In addition, electron microscopy revealed the presence of virus-like particles in the cytoplasm, and a considerable number of viruses were budding from the cell membrane of the jejunal EIEs. These findings indicate that the viruses replicated inside the cell and were released from the cell membrane into the extracellular space.Thus, ECoV can infect EIEs derived from all three regions of the small intestine and substantially replicate. To our knowledge, this is the first report describing enteric virus infection in EIEs. Our previous experimental challenge study in horses showed that ECoV infected throughout the small and large intestinal tissues, as confirmed by RT-qPCR and \u003cem\u003ein situ\u003c/em\u003e hybridization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The current study reproduced the \u003cem\u003ein vivo\u003c/em\u003e situation, showing that EIEs offer a promise as a novel \u003cem\u003ein vitro\u003c/em\u003e model for studying ECoV infection. In humans and other animals, the potential applications of intestinal enteroids include the identification of the target cells of enteric pathogens, the elucidation of infectious mechanisms, and the isolation of viruses from clinical specimen [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To advance these studies in EIEs, further research is needed.\u003c/p\u003e \u003cp\u003eOne limitation of the current study is that EIEs were generated from the tissues of only one horse. It is important to note that the intestinal enteroids originate from the donor\u0026rsquo;s tissues and therefore may exhibit characteristics inherited from those tissues. It is probable that enteroids derived from different individuals differ in susceptibility to enteric pathogens. For example, the growth rate of human rotavirus differed among intestinal enteroids derived from individual humans [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, it is preferable to generate EIEs from multiple individuals and compare their capacity to sustain viral infection in order to confirm whether our results are representative of EIEs in general.\u003c/p\u003e \u003cp\u003eIn conclusion, we generated duodenal, jejunal, and ileal enteroids from horse tissues and demonstrated that ECoV infects and replicates in these enteroids in a 2D monolayer model. EIEs offer promise as a robust tool with which to investigate interactions between the equine intestinal epithelium and ECoV \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol and all animal procedures were approved by the Animal Care Committee of the Equine Research Institute of the Japan Racing Association with approved numbers 21-6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed in the study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur research was funded by the Japan Racing Association.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYK participated in all experiments and drafted the manuscript. MN and TS contributed to design and supervision of this study. AO, DK, and TU performed all histological analyses. KT supported RT-qPCR analysis. HB, MO, and NK reviewed the manuscript and provided advice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNC99 was provided by James S. Guy, a professor of veterinary virology at North Carolina University, in 2001. We are grateful to Akira Kokubun, Akiko Kasagawa, Miwa Tanaka, and Kaoru Watanabe (Equine Research Institute, Japan Racing Association) for their invaluable technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKambayashi Y, Kishi D, Ueno T, Ohta M, Bannai H, Tsujimura K, Kinoshita Y, Nemoto M (2022) Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses. Arch Virol 167:1611\u0026ndash;1618\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKambayashi Y, Bannai H, Tsujimura K, Hirama A, Ohta M, Nemoto M (2021) Outbreak of equine coronavirus infection among riding horses in Tokyo, Japan, Comp Immunol Microbiol Infect Dis. 77:101668\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFielding CL, Higgins JK, Higgins JC, McIntosh S, Scott E, Giannitti F, Mete A, Pusterla N (2015) Disease associated with equine coronavirus infection and high case fatality rate. J Vet Intern Med 29:307\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiannitti F, Diab S, Mete A, Stanton JB, Fielding L, Crossley B, Sverlow K, Fish S, Mapes S, Scott L, Pusterla N (2015) Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids, Vet Pathol. 52:1148\u0026ndash;1156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuy JS, Breslin JJ, Breuhaus B, Vivrette S, Smith LG (2000) Characterization of a coronavirus isolated from a diarrheic foal. J Clin Microbiol 38:4523\u0026ndash;4526\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOue Y, Morita Y, Kondo T, Nemoto M (2013) Epidemic of equine coronavirus at Obihiro Racecourse, Hokkaido, Japan in 2012. J Vet Med Sci 75:1261\u0026ndash;1265\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOue Y, Ishihara R, Edamatsu H, Morita Y, Yoshida M, Yoshima M, Hatama S, Murakami K, Kanno T (2011) Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain. Vet Microbiol 150:41\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEttayebi K, Crawford SE, Murakami K, Broughman JR, Karandikar U, Tenge VR, Neill FH, Blutt SE, Zeng XL, Qu L, Kou B, Opekun AR, Burrin D, Graham DY, Ramani S, Atmar RL, Estes MK (2016) Replication of human noroviruses in stem cell-derived human enteroids. Science 353:1387\u0026ndash;1393\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L, Fu F, Guo S, Wang H, He X, Xue M, Yin L, Feng L, Liu P (2019) Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response. J Virol. 93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Li C, Liu X, Chiu MC, Zhao X, Wang D, Wei Y, Lee A, Zhang AJ, Chu H, Cai JP, Yip CC, Chan IH, Wong KK, Tsang OT, Chan KH, Chan JF, To KK, Chen H, Yuen KY (2020) Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med 26:1077\u0026ndash;1083\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTekes G, Ehmann R, Boulant S, Stanifer ML (2020) Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection, Cells. 9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262\u0026ndash;265\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141:1762\u0026ndash;1772\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart AS, Freund JM, Gonzalez LM (2018) Advanced three-dimensional culture of equine intestinal epithelial stem cells. Equine Vet J 50:241\u0026ndash;248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellman S (2021) Generation of equine enteroids and enteroid-derived 2D monolayers that are responsive to microbial mimics. Vet Res 52:108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellman S, Martin F, Tyd\u0026eacute;n E, Sellin ME, Norman A, Hjertner B, Svedberg P, Fossum C (2024) Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection. Vet Res 55:25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePusterla N, Mapes S, Wademan C, White A, Ball R, Sapp K, Burns P, Ormond C, Butterworth K, Bartol J, Magdesian KG (2013) Emerging outbreaks associated with equine coronavirus in adult horses. Vet Microbiol 162:228\u0026ndash;231\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWindhaber C, Heckl A, Csukovich G, Pratscher B, Burgener IA, Biermann N, Dengler F (2024) A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium. Vet Res 55:30\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Li C, Liu X, Chiu MC, Wang D, Wei Y, Chu H, Cai JP, Hau-Yee Chan I, Kak-Yuen Wong K, Fuk-Woo Chan J, Kai-Wang To K, Yuen KY, Zhou J (2021) Human Intestinal Organoids Recapitulate Enteric Infections of Enterovirus and Coronavirus. Stem Cell Rep 16:493\u0026ndash;504\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoodsant T, Navis M, Aknouch I, Renes IB, van Elburg RM, Pajkrt D, Wolthers KC, Schultsz C, van der Ark KCH, Sridhar A, Muncan V (2020) A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine. Front Cell Infect Microbiol 10:272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Hee B, Loonen LMP, Taverne N, Taverne-Thiele JJ, Smidt H, Wells JM (2018) Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids. Stem Cell Res 28:165\u0026ndash;171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxena K, Blutt SE, Ettayebi K, Zeng XL, Broughman JR, Crawford SE, Karandikar UC, Sastri NP, Conner ME, Opekun AR, Graham DY, Qureshi W, Sherman V, Foulke-Abel J (2016) In J., Kovbasnjuk O., Zachos N.C., Donowitz M., Estes M.K., Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology, J Virol. 90:43\u0026ndash;56\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"equine coronavirus, equine intestinal enteroid, horse, viral infection","lastPublishedDoi":"10.21203/rs.3.rs-4540308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4540308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as \u003cem\u003ein vitro\u003c/em\u003e models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed \u003cem\u003ein vivo\u003c/em\u003e in the intestinal epithelium. qRT-PCR and electron microscopy showed that ECoV had infected and replicated in the EIEs. These results suggest that EIEs can be novel \u003cem\u003ein vitro\u003c/em\u003e tools for studying the interaction between equine intestinal epithelium and ECoV. (88 words)\u003c/p\u003e","manuscriptTitle":"Equine coronavirus infection and replication in equine intestinal enteroids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 16:30:05","doi":"10.21203/rs.3.rs-4540308/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9175547a-2726-461c-8d1e-5d8dcaffa4ad","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:04:59+00:00","versionOfRecord":{"articleIdentity":"rs-4540308","link":"https://doi.org/10.1186/s13567-024-01381-z","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2024-10-10 15:57:23","publishedOnDateReadable":"October 10th, 2024"},"versionCreatedAt":"2024-07-03 16:30:05","video":"","vorDoi":"10.1186/s13567-024-01381-z","vorDoiUrl":"https://doi.org/10.1186/s13567-024-01381-z","workflowStages":[]},"version":"v1","identity":"rs-4540308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4540308","identity":"rs-4540308","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}