Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2 + mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with COVID-19. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression. RESULTS SARS-CoV-2 infects human intestinal enteroidsIn the intestine, ACE2 functions as a chaperone for the sodiumdependent neutral amino acid transporter B 0 AT1 (encoded by SLC6A19) on IECs and regulates microbial homeostasis (21,22). ACE2 expression is substantially higher in the small intestine than in all the other organs, including the lung, in both humans and mice (fig. S1, A and B). Given these data, we assessed whether SARS-CoV-2 can infect IECs as a first step for understanding its implications for fecal-oral transmission. We performed single-cell RNA sequencing (scRNA-seq) to capture the global transcriptomics in all IEC subsets in the mouse small intestinal epithelium (Fig. 1A, left). ACE2 mRNA was predominantly seen in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes (Fig. 1A, right) (23, 24). In addition, bulk RNA-seq revealed that primary human ileum enteroids had substantially higher mRNA levels of all known CoV receptors, including ACE2, than the of 10 that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 10 5 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 10 5 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were measured using a TCID 50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, ...

Highlights d Vesicular stomatitis virus encoding the SARS-CoV-2 spike replicates to high titers d Virus propagation is enhanced by a truncation in the cytoplasmic tail of the spike d Neutralization can be assessed by BSL2 and BSL3 highthroughput assays d SARS-CoV-2-and VSV-SARS-CoV-2-based neutralization assays correlate

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

25Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and 26 possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the 27 level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus 28 as to which assay should be used for such measurements. Using an infectious molecular clone 29 of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the 30 glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and 31 developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also 32 developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety 33 level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody 34 preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally 35 high degree of concordance. The two assays will help define correlates of protection for antibody-36 based countermeasures including therapeutic antibodies, immune γ-globulin or plasma 37 preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-38

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Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Two compounds, nitazoxanide and JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 4.90 μM and 0.69 μM, respectively, with specificity indices of greater than 150. Both inhibitors had in vitro antiviral activity in multiple cell types against some DNA and RNA viruses, including porcine transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved body weight gain and survival. These results highlight the potential utility of nitazoxanide and JIB-04 as antiviral agents against SARS-CoV-2 and other viral pathogens.

24Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows 25 broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG 26 screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified 27 CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of 28 virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes 29 and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed 30 membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and 31 provide the molecular basis for its possible therapeutic development. 32 33 Main Text 34The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the 35 etiological agent of coronavirus disease-2019 (COVID-19) 1, 2 , has infected millions of 36 people worldwide and caused hundreds of thousands of deaths, with a case fatality rate 37 as high as 5% 3 . Currently, there are no FDA approved vaccines available. In most 38 instances, treatment is limited to supportive therapies to help alleviate symptoms. 39Chloroquine phosphate, hydroxychloroquine sulfate, and a polymerase inhibitor 40 remdesivir represent the only drug products that the FDA has approved for emergency 41 use authorization 4 , and concern exists that monotherapy would rapidly result in the 42 emergence of resistance. There is a pressing need to identify effective antivirals as 43 countermeasures before safe and efficacious vaccines are developed and deployed. 44Here, we sought to harness the host innate immune responses to inhibit SARS-CoV-2 45 replication. Interferons (IFNs) are a group of small, secreted proteins 5, 6 that potently 46 clinical isolate of SARS-CoV-2 also was suppressed in HEK293-hACE2 cells expressing 92 CH25H compared to control plasmid transfection (Fig. 1E). 93 94 Next, we tested whether the antiviral activity of CH25H depends on 25HC synthesis. As 95 compared to the control 7-α, 25-OHC, pre-treatment of HEK293-hACE2 cells with 25HC 96 for 1 hour prior to VSV-SARS-CoV-2 infection recapitulated the suppressive effect of 97 CH25H overexpression and reduced virus replication ( Fig. 2A). 25HC dose-dependently 98 inhibited VSV-SARS-CoV-2 infection in MA104 cells, with an approximate EC50 of 1.03 99 µM (Fig. 2B). No cytotoxicity was observed at the highest concentration tested (30 µM). 100When plaque assays were performed in the presence of 25HC, there was a reduction in 101 both plaque numbers and sizes (Fig. S2A-B). Wild-type SARS-CoV-2 virus replication 102 also was inhibited by 25HC but not 7-α, 25-OHC treatment (Fig. 2C). Collectively, our 103 results suggest an antiviral activity of CH25H and its natural product 25HC in suppressing 104 SARS-CoV-2 virus infection. 105 106During SARS-CoV-2 entry into host cells, S protein binding to ACE2 enables its cleavage 107 by membrane-bound TMPRSS serine proteases and subsequent fusion of the viral 108 membrane to the host cell membrane 17, 29, 30 . Previous work suggests that...