SARS-CoV-2 productively infects human gut enterocytes

The virus severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission via the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2 as demonstrated by confocal- and electron-microscopy. Consequently, significant titers of infectious viral particles were detected. mRNA expression analysis revealed strong induction of a generic viral response program. Hence, intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology

S evere acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, emerged in 2003 (1). In late 2019, a novel transmissible coronavirus, SARScoronavirus 2 (SARS-CoV-2), was noted to cause an influenza-like disease ranging from mild respiratory symptoms to severe lung injury, multiorgan failure, and death (2)(3)(4). SARS-CoV and SARS-CoV-2 belong to the Sarbecovirus subgenus of the genus Betacoronavirus in the family Coronaviridae (5)(6)(7). The SARS-CoV receptor is angiotensin-converting enzyme 2 (ACE2) (8,9). The spike proteins of both viruses bind to ACE2, whereas soluble ACE2 blocks infection by SARS-CoV and SARS-CoV-2 (10)(11)(12)(13). Transmission of SARS-CoV-2 is thought to occur through respiratory droplets and fomites. The virus can be detected in upper respiratory tract samples, implicating the nasopharynx as a site of replication. In human lung, ACE2 is expressed mainly in alveolar epithelial type II cells and ciliated cells (14)(15)(16). However, the highest expression of ACE2 in the human body occurs in the brush border of intestinal enterocytes (14,17). Even though respiratory symptoms dominate the clinical presentation of COVID-19, gastrointestinal symptoms are observed in a subset of patients (18,19). Moreover, viral RNA can be found in rectal swabs even after nasopharyngeal testing has turned negative, implying gastrointestinal infection and a fecal-oral transmission route (20)(21)(22).

SARS-CoV-2 infects airway and gut organoids
Organoids are three-dimensional (3D) structures that can be grown from adult stem cells and recapitulate key aspects of the organ from which those cells derive. Because SARS-CoV and SARS-CoV-2 target the lung, we added virus to organoid-derived human airway epithelium cultured in 2D and observed that SARS-CoV and SARS-CoV-2 readily infected differentiated airway cultures. (Fig. 1A). Immunostaining reveal that the viruses targeted ciliated cells but not goblet cells (Fig. 1, B and C).
Human small intestinal organoids (hSIOs) are established from primary gut epithelial stem cells, can be expanded indefinitely in 3D culture, and contain all proliferative and differentiated cell types of the in vivo epithelium (23). hSIOs have also allowed the first in vitro culturing of norovirus (24). We exposed ileal hSIOs grown under four different culture conditions (EXP, DIF, DIF-BMP, and EEC) to SARS-CoV and SARS-CoV-2 at a multiplicity of infection of 1. hSIOs grown in Wnt highexpansion (EXP) medium overwhelmingly consisted of stem cells and enterocyte progenitors. Organoids grown in differentiation (DIF) medium contained enterocytes, goblet cells, and low numbers of enteroendocrine cells (EECs). The addition of BMP2/4 to DIF medium (DIF-BMP medium) led to further maturation (25). In the final condition (EEC), we induced the expression of NeuroG3 from a stably transfected vector with doxycycline to raise EEC numbers ( fig. S3D). Samples were harvested at multiple time points after infection and processed for the analyses shown in Figs. 2 to 5. Both SARS-CoV and SARS-CoV-2 productively infected hSIOs, as assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) for viral sequences and by live virus titrations on VeroE6 cells (see Fig. 2 for lysed organoids and fig. S1 for organoid supernatant). Infectious virus particles and viral RNA increased for both viruses in all conditions. Because EXP medium supported virus replication (Fig. 2, A and E), enterocyte progenitors appeared to be a primary viral target. Differentiated organoids (grown in DIF and DIF-BMP medium) produced slightly (nonstatistically significant) lower levels of *These authors contributed equally to this work. †These authors contributed equally to this work. infectious virus ( Fig. 2 and fig. S1). In organoids induced to generate EECs, virus yields were similar to those in EXP medium (Fig. 2, D and H). In differentiated hSIOs, SARS-CoV-2 titers remained stable at 60 hours after infection, whereas SARS-CoV titers dropped by 1 to 2 log (Fig. 2, B, C, F, and G). The latter decline was not observed in infected hSIOs grown in EXP medium. Culture supernatants across culture conditions contained lower levels of infectious virus compared with lysed hSIOs, implying that virus was primarily secreted apically (fig. S1, A to D). Despite this, viral RNA was detected readily in culture supernatants, correlating with the infectious virus levels within hSIOs (Fig. 2, E to H, and fig. S1, E to H). ACE2 mRNA expression differed greatly between the four conditions. EXP-hSIOs expressed 300-fold less ACE2 mRNA compared with DIF-hSIOs when analyzed in bulk ( fig. S2). BMP treatment induced 6.5-fold up-regulation of ACE2 mRNA compared with DIF treatment alone. Because this did not yield infection rate differences, the DIF-BMP condition was not analyzed further.

SARS-CoV-2 infects enterocyte lineage cells
To determine the target cell type, we then performed confocal analysis on hSIOs cultured in EXP, DIF, or EEC conditions. We stained for viral double-stranded RNA (dsRNA), viral nucleocapsid protein, KI67 to visualize proliferative cells, actin (using phalloidin) to visualize enterocyte brush borders, and DNA (DAPI) and cleaved caspase 3 to visualize apoptotic cells. Generally, comparable rates of viral in-fections were observed in the organoids growing in all three conditions. We typically noted staining for viral components (white) in rare, single cells at 24 hours. At 60 hours, the number of infected cells had substantially increased (Fig. 3A). Infected cells invariably displayed proliferative enterocyte progenitor phenotypes (EXP; Fig. 3B, top) or ApoA1 + enterocyte phenotypes (DIF; Fig. 3B, bottom). SARS-CoV also readily infected enterocyte lineage cells ( fig.  S3, A and B), as was shown previously (26,27).  . S5). ACE2 protein was readily revealed as a bright and ubiquitous brush border marker in hSIOs in DIF medium (Fig. 3C). In hSIOs in EXP medium, ACE2 staining was much lower, yet still apical, in occasional cells in a subset of organoids that displayed a more mature morphology (Fig. 3C). In immature (cystic) organoids within the same cultures, the ACE2 signal was below the detection threshold. The percentages of infected organoids under EXP and DIF conditions are given in fig. S4. Figure S5 shows images and quantification of apoptotic cells upon infection.

Ultrastructural analysis of the viral life cycle in enterocytes
Unsupervised transmission electron microscopy (28) was performed on selected highly infected samples. Figure 4 shows two hSIOs selected from 42 hSIOs imaged at 60 hours after SARS-CoV-2 infection. These differ in the state of infection: Whereas the cellular organization within organoid 1 was still intact  (Fig. 4I) at the basolateral (Fig. 4J) and apical side (Fig.  4K) of enterocytes. Double-membrane vesicles, which are the subcellular site of viral replication (29), are visualized in Fig. 4, E and P. The nuclei in both organoids differed from nuclei in mock-infected organoids by having a slightly rounder shape. Other differences were that the nuclear contour index (30) was 4.0 ± 0.5 versus 4.3 ± 0.5 for the control set, and there was more heterochromatin (Fig. 4N) and one or two dense nucleoli in the center (Fig. 4O).

RNA expression changes in infected enterocytes
We then performed mRNA-sequencing analysis to determine gene expression changes induced by SARS-CoV and SARS-CoV-2-infection of hSIOs cultured continuously in EXP medium and hSIOs cultured in DIF medium. Infection with SARS-CoV-2 elicited a broad signature of cytokines and interferon (IFN)-stimulated genes (ISGs) attributed to type I and III IFN responses ( Fig. 5A and tables S1 and S2), as confirmed by gene ontology analysis (Fig. 5B). An overlapping list of genes appeared in SARS-CoV-2-infected DIF organoids ( fig. S6 and table  S3). mRNA-sequencing analysis confirmed differentiation of DIF organoids into multiple intestinal lineages, including ACE2 up-regulation ( fig. S7). SARS-CoV also induced ISGs but to a much lower level (table S4). Figure 5C shows the regulation of SARS-CoV-2-induced genes in SARS-CoV-infected organoids. This induction was similar to infections with other viruses such as norovirus (31), rotavirus (32), and enteroviruses (33,34). A recent study (35) described an antiviral signature induced in human cell lines after SARS-CoV-2 infection. Whereas the ISG response was broader in hSIOs, the induced gene sets were in close agreement between the two datasets ( fig. S8). One obvious similarity was the low expression of type I and III IFNs: We only noticed a small induction of the type III IFN IFNL1 in SARS-CoV-2-infected organoids. In SARS-CoVinfected organoids, we did not observe any type I or type III IFN induction. We confirmed these findings by enzyme-linked immunosorbent assay (ELISA) on the culture supernatant and qRT-PCR on extracted RNA of the hSIOs, which in addition to IFNL1, picked up low levels of type I IFN IFNB1 in SARS-CoV-2-but not in SARS-CoV-infected organoids ( fig. S9). The specific induction of IP-10/CXCL10 and ISG15 by SARS-CoV-2 was also confirmed by ELISA and qRT-PCR, respectively (fig. S10). As in a previous study (35), a few cytokine genes were induced by both viruses, albeit to modest levels. For a comparison with (35), see fig. S11. Altogether, these data indicate that SARS-CoV-2 induces a stronger IFN response than SARS-CoV in hSIOs. Finally, the infection was repeated in a second experiment in the same ileal hSIO line and analyzed after 72 hours. Analysis involved viral titration ( fig. S12), confocal imaging (fig. S13), and mRNA sequencing ( fig. S14). This experiment essentially confirmed the observations presented above. A limited, qualitative experiment applying confocal analysis demonstrated the infectability of two other lines available in the laboratory (one ileal and one duodenal) from independent donors ( fig. S13). This study shows that SARS-CoV and SARS-CoV-2 infect enterocyte lineage cells in an hSIO model. We observed similar infection rates of enterocyte precursors and enterocytes, whereas ACE2 expression increased~1000-fold upon differentiation at the mRNA level ( fig.  S2). This suggests that low levels of ACE2 may be sufficient for viral entry.
SARS-CoV-2 is the third highly pathogenic coronavirus (after SARS-CoV and MERS-CoV) to jump to humans within <20 years, suggesting that new zoonotic coronavirus spillovers are likely to occur in the future. Despite this, limited information is available on coronavirus pathogenesis and transmission, in part because of the lack of in vitro cell models that accurately model host tissues. Very recently, it was shown that human induced pluripotent stem cells differentiated toward a kidney fate supported replication of SARS-CoV-2 (13). Our data suggest that human organoids represent faithful experimental models with which to study the biology of coronaviruses.   IFI6  IFI27  IFITM1  MX1  RN7SK  ARC  DKK1  MT1G  SAMD9  IFIT3  C10orf99  IFIT2  NKX3-1  LINC00941  HSPA6  IFIT1  IFI44L  CMPK2  RSAD2  ISG15  OAS2  CXCL11  IFI44  CXCL10 IFI6  IFI27  IFITM1  MX1  RN7SK  ARC  DKK1  MT1G  SAMD9  IFIT3  C10orf99  IFIT2  NKX3-1  LINC00941  HSPA6  IFIT1  IFI44L  CMPK2  RSAD2  ISG15  OAS2  CXCL11  IFI44  CXCL10  SLC34A2