Lung Organoids as Model to Study SARS-CoV-2 Infection.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has severely affected socio-economic conditions and people's life. The lung is the major target organ infected and (seriously) damaged by SARS-CoV-2, so a comprehensive understanding of the virus and the mechanism of infection are the first choices to overcome COVID-19. Recent studies have demonstrated the enormous value of human organoids as platforms for virological research, making them an ideal tool for researching host-pathogen interactions. In this study, the various existing lung organoids and their identification biomarkers and applications are summarized. At the same time, the seven coronaviruses currently capable of infecting humans are outlined. Finally, a detailed summary of existing studies on SARS-CoV-2 using lung organoids is provided and includes pathogenesis, drug development, and precision treatment. This review highlights the value of lung organoids in studying SARS-CoV-2 infection, bringing hope that research will alleviate COVID-19-associated lung infections.

Human Nasal Organoids Model SARS-CoV-2 Upper Respiratory Infection and Recapitulate the Differential Infectivity of Emerging Variants.

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Applications of Brain Organoids for Infectious Diseases.

Brain organoids are self-organized three-dimensional aggregates generated from pluripotent stem cells. They exhibit complex cell diversities and organized architectures that resemble human brain development ranging from neural tube formation, neuroepithelium differentiation, neurogenesis and gliogenesis, to neural circuit formation. Rapid advancements in brain organoid culture technologies have allowed researchers to generate more accurate models of human brain development and neurological diseases. These models also allow for direct investigation of pathological processes associated with infectious diseases affecting the nervous system. In this review, we first briefly summarize recent advancements in brain organoid methodologies and neurodevelopmental processes that can be effectively modeled by brain organoids. We then focus on applications of brain organoids to investigate the pathogenesis of neurotropic viral infection. Finally, we discuss limitations of the current brain organoid methodologies as well as applications of other organ specific organoids in the infectious disease research.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry.

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Adult stem cell-derived complete lung organoid models emulate lung disease in COVID-19.

Background: SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. Methods: We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Results: Infected ALO monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Conclusions: Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. Funding: This work was supported by the National Institutes for Health (NIH) grants 1R01DK107585-01A1, 3R01DK107585-05S1 (to SD); R01-AI141630, CA100768 and CA160911 (to PG) and R01-AI 155696 (to PG, DS and SD); R00-CA151673 and R01-GM138385 (to DS), R01- HL32225 (to PT), UCOP-R00RG2642 (to SD and PG), UCOP-R01RG3780 (to P.G. and D.S) and a pilot award from the Sanford Stem Cell Clinical Center at UC San Diego Health (P.G, S.D, D.S). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. L.C.A's salary was supported in part by the VA San Diego Healthcare System. This manuscript includes data generated at the UC San Diego Institute of Genomic Medicine (IGC) using an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929).

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Advancing lung organoids for COVID-19 research.

The COVID-19 pandemic has emphasised the need to develop effective treatments to combat emerging viruses. Model systems that poorly represent a virus' cellular environment, however, may impede research and waste resources. Collaborations between cell biologists and virologists have led to the rapid development of representative organoid model systems to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that lung organoids, in particular, have advanced our understanding of SARS-CoV-2 pathogenesis, and have laid a foundation to study future pandemic viruses and develop effective treatments.

Experimental Models for SARS-CoV-2 Infection.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a novel virus that causes coronavirus disease 2019 (COVID-19). To understand the identity, functional characteristics and therapeutic targets of the virus and the diseases, appropriate infection models that recapitulate the in vivo pathophysiology of the viral infection are necessary. This article reviews the various infection models, including Vero cells, human cell lines, organoids, and animal models, and discusses their advantages and disadvantages. This knowledge will be helpful for establishing an efficient system for defense against emerging infectious diseases.

Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.

Review of studies of severe acute respiratory syndrome related coronavirus-2 pathogenesis in human organoid models.

Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) is the cause of Covid-19 which was classified as a global pandemic in March 2020. The increasing global health and economic burden of SARS-CoV-2 has necessitated urgent investigations into the pathogenesis of disease and development of therapeutic and vaccination regimens. Human trials of vaccine and antiviral candidates have been undertaken, but basic pathogenetic studies are still required to inform these trials. Gaps in understanding of cellular infection by, and immunity to, SARS-CoV-2 mean additional models are required to assist in improved design of these therapeutics. Human organoids are three-dimensional models that contain multiple cell types and mimic human organs in ex vivo culture conditions. The SARS-CoV-2 virus has been implicated in causing not only respiratory injury but also injury to other organs such as the brain, liver and kidneys. Consequently, a variety of different organoid models have been employed to investigate the pathogenic mechanisms of disease due to SARS-CoV-2. Data on these models have not been systematically assembled. In this review, we highlight key findings from studies that have utilised different human organoid types to investigate the expression of SARS-CoV-2 receptors, permissiveness, immune response, dysregulation of cellular functions, and potential antiviral therapeutics.

The Organoid Platform: Promises and Challenges as Tools in the Fight against COVID-19.

Many pathogenic viruses that affect man display species specificity, limiting the use of animal models. Studying viral biology and identifying potential treatments therefore benefits from the development of in vitro cell systems that closely mimic human physiology. In the current COVID-19 pandemic, rapid scientific insights are of the utmost importance to limit its impact on public health and society. Organoids are emerging as versatile tools to progress the understanding of SARS-CoV-2 biology and to aid in the quest for novel treatments.

SARS-CoV-2 Infection and Disease Modelling Using Stem Cell Technology and Organoids.

In this Review, we briefly describe the basic virology and pathogenesis of SARS-CoV-2, highlighting how stem cell technology and organoids can contribute to the understanding of SARS-CoV-2 cell tropisms and the mechanism of disease in the human host, supporting and clarifying findings from clinical studies in infected individuals. We summarize here the results of studies, which used these technologies to investigate SARS-CoV-2 pathogenesis in different organs. Studies with in vitro models of lung epithelia showed that alveolar epithelial type II cells, but not differentiated lung alveolar epithelial type I cells, are key targets of SARS-CoV-2, which triggers cell apoptosis and inflammation, while impairing surfactant production. Experiments with human small intestinal organoids and colonic organoids showed that the gastrointestinal tract is another relevant target for SARS-CoV-2. The virus can infect and replicate in enterocytes and cholangiocytes, inducing cell damage and inflammation. Direct viral damage was also demonstrated in in vitro models of human cardiomyocytes and choroid plexus epithelial cells. At variance, endothelial cells and neurons are poorly susceptible to viral infection, thus supporting the hypothesis that neurological symptoms and vascular damage result from the indirect effects of systemic inflammatory and immunological hyper-responses to SARS-CoV-2 infection.