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Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses., Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Here the authors generate mutant clonal intestinal organoids for 19 host genes previously implicated in coronavirus biology and identify the cell surface protease TMPRSS2 as a potential therapeutic target.

A new phase of the COVID-19 pandemic has started as several SARS-CoV-2 variants are rapidly emerging globally, raising concerns for increased transmissibility. As animal models and traditional in vitro systems may fail to model key aspects of the SARS-CoV-2 replication cycle, representative in vitro systems to assess variants phenotypically are urgently needed. We found that the British variant (clade B.1.1.7), compared to an ancestral SARS-CoV-2 clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil human organoids as powerful tools to phenotype viral variants and suggest extended shedding as a correlate of fitness for SARS-CoV-2.One-Sentence SummaryBritish SARS-CoV-2 variant (clade B.1.1.7) infects organoids for extended time and has a higher fitness in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air–liquid interface culture system which was characterized by confocal and electron microscopy and single‐cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self‐renewing fetal lung bud tip organoids. These cultures were readily infected by SARS‐CoV‐2 with mainly surfactant protein C‐positive alveolar type II‐like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS‐CoV‐2 infection and can be applied for drug screens., A human airway in vitro culture permissive to COVID‐19 demonstrates a drug‐sensitive IFN response.

After the SARS-CoV outbreak in 2003, a second zoonotic coronavirus named SARS-CoV-2, emerged late 2019 in China and rapidly caused the COVID-19 pandemic leading to a public health crisis of an unprecedented scale. Despite the fact that SARS-CoV-2 uses the same receptor as SARS-CoV, transmission and pathogenesis of both viruses seem to be quite distinct. A remarkable feature of the SARS-CoV-2 spike is the presence of a multibasic cleavage site, which is absent in the SARS-CoV spike. The viral spike protein not only attaches to the entry receptor, but also mediates fusion after cleavage by host proteases. Here, we report that the SARS-CoV-2 spike multibasic cleavage site increases infectivity on differentiated organoid-derived human airway cells. Compared with SARS-CoV, SARS-CoV-2 entered faster into the lung cell line Calu-3, and more frequently formed syncytial cells in differentiated organoid-derived human airway cells. Moreover, the multibasic cleavage site increased entry speed and plasma membrane serine protease usage relative to endosomal entry using cathepsins. Blocking serine protease activity using the clinically approved drug camostat mesylate effectively inhibited SARS-CoV-2 entry and replication in differentiated organoid-derived human airway cells. Our findings provide novel information on how SARS-CoV-2 enters relevant airway cells and highlight serine proteases as an attractive antiviral target.Significance StatementHighly pathogenic coronaviruses have spilled from animals to humans three times in the past two decades. Late 2019, SARS-CoV-2 emerged in China and was declared a pandemic by March 2020. The other two highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, emerged in 2002 and 2012, respectively, but did not attain sustained human-to-human transmission. Given the high diversity of coronaviruses in animals, urbanization and increased air travel, future coronavirus pandemics are likely to occur intermittently. Identifying which factors determine pandemic potential and pathogenicity are therefore of key importance to global health. Additionally, there is an urgent need to rapidly translate fundamental knowledge to the clinic, a process that is expedited through the use of relevant cell culture systems.

Intestinal organoids as an infection model Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an influenza-like disease with a respiratory transmission route; however, patients often present with gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain. Moreover, the virus has been detected in anal swabs, and cells in the inner-gut lining express the receptor that SARS-CoV-2 uses to gain entry to cells. Lamers et al. used human intestinal organoids, a “mini-gut” cultured in a dish, to demonstrate that SARS-CoV-2 readily replicates in an abundant cell type in the gut lining—the enterocyte—resulting in the production of large amounts of infective virus particles in the intestine. This work demonstrates that intestinal organoids can serve as a model to understand SARS-CoV-2 biology and infectivity in the gut. Science , this issue p. 50