Exploring host-commensal-pathogen dynamics in cell line and organotypic human intestinal epithelial models.

Host and microbiome intricately interact in the ecosystem of the human digestive tract, playing a crucial role in our health. These interactions can initiate immune responses in the epithelial cells, which, in turn, activate downstream responses in other immune cells. Here, we used a CaCo-2 and a human intestinal enteroid (HIE) model to explore epithelial responses to both commensal and pathogenic bacteria, individually and combined. CaCo-2 cells were co-cultured with peripheral blood mononuclear cells, revealing downstream activation of immune cells. While both systems showed comparable cytokine profiles, they differed in their responses to the different bacteria, with the organoid system being more representative of responses observed in humans. We provide evidence of the pro-inflammatory responses associated with these bacteria. These models contribute to a deeper understanding of the interactions between the microbiota, intestinal epithelium, and immune cells in the gut, promoting advances in the field of host-microbe interactions.

Parechovirus infection in human brain organoids: host innate inflammatory response and not neuro-infectivity correlates to neurologic disease.

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease, the highly prevalent PeV-A1 is not associated with CNS disease. Here, we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively, our findings align with clinical observations and suggest a role for neuroinflammation, rather than viral replication, in PeV-A3 (and E11) infection.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Assessment of the broad-spectrum host targeting antiviral efficacy of halofuginone hydrobromide in human airway, intestinal and brain organotypic models.

Halofuginone hydrobromide has shown potent antiviral efficacy against a variety of viruses such as SARS-CoV-2, dengue, or chikungunya virus, and has, therefore, been hypothesized to have broad-spectrum antiviral activity. In this paper, we tested this broad-spectrum antiviral activity of Halofuginone hydrobomide against viruses from different families (Picornaviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, and Flaviviridae). To this end, we used relevant human models of the airway and intestinal epithelium and regionalized neural organoids. Halofuginone hydrobomide showed antiviral activity against SARS-CoV-2 in the airway epithelium with no toxicity at equivalent concentrations used in human clinical trials but not against any of the other tested viruses.

First description and phylogenetic analysis of coxsackie virus A non-polio enteroviruses and parechoviruses A in South Sudanese children.

Enteroviruses (EV) and parechoviruses A (PeV-A) are commonly circulating viruses able to cause severe disease. Surveillance studies from sub-Saharan Africa are limited and show high but variable infection rates and a high variation in genotypes. This is the first study to describe EV and PeV-A circulation in children in South Sudan. Of the fecal samples collected, 35% and 10% were positive for EV and PeV-A, respectively. A wide range of genotypes were found, including several rarely described EV and PeV-A types. Coxsackie virus A (CVA) EV-C types, particularly CVA13, were the most dominant EV types. The CVA13 types had a high diversity with the majority belonging to four different previously described clusters. PeV-A1 and -A14 were the most common PeV-A genotypes. A lack of representative data from our and other studies from sub-Saharan Africa demonstrates the need for more systematic surveillance of non-polio EV and PeV-A types in this region.

What is a neurotropic virus: Discrepancies in terminology between clinical and basic science.

Technological advancements allow for the use of more physiologically relevant models to study viral neuropathology. This results in closure of the gap between clinical and basic research. We discuss the current discrepancy in the use of terminology around viral CNS infections, which impedes interdisciplinary communication and translation of findings.

Non-Polio Enterovirus C Replicate in Both Airway and Intestine Organotypic Cultures.

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99.

When science meets entrepreneurship.

Science industries, such as the health and medical industry, are experiencing increases in competition regarding commercializing, patenting, and funding of scientific outputs. As such, scientists are facing increased expectation to engage in academic entrepreneurship. OrganoVIR (Organoids for Virus Research) is a Horizon2020 Innovative Training Network (ITN) that aims to train Early Stage Researchers (ESRs) to lead innovation in the field of human organoids for virus research. To assist them in this process, OrganoVIR introduced a pre-Master of Business Administration program that introduced OrganoVIR's ESRs to the tools and knowledge necessary to navigate the competitive industry. In this article, we describe this innovative pre-Master of Business Administration program and highlight the importance as well as the need for having a pre-Master of Business Administration programs in a scientific training network.

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection. Importance: With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

A beginner's guide on the use of brain organoids for neuroscientists: a systematic review.

BACKGROUND: The first human brain organoid protocol was presented in the beginning of the previous decade, and since then, the field witnessed the development of many new brain region-specific models, and subsequent protocol adaptations and modifications. The vast amount of data available on brain organoid technology may be overwhelming for scientists new to the field and consequently decrease its accessibility. Here, we aimed at providing a practical guide for new researchers in the field by systematically reviewing human brain organoid publications. METHODS: Articles published between 2010 and 2020 were selected and categorised for brain organoid applications. Those describing neurodevelopmental studies or protocols for novel organoid models were further analysed for culture duration of the brain organoids, protocol comparisons of key aspects of organoid generation, and performed functional characterisation assays. We then summarised the approaches taken for different models and analysed the application of small molecules and growth factors used to achieve organoid regionalisation. Finally, we analysed articles for organoid cell type compositions, the reported time points per cell type, and for immunofluorescence markers used to characterise different cell types. RESULTS: Calcium imaging and patch clamp analysis were the most frequently used neuronal activity assays in brain organoids. Neural activity was shown in all analysed models, yet network activity was age, model, and assay dependent. Induction of dorsal forebrain organoids was primarily achieved through combined (dual) SMAD and Wnt signalling inhibition. Ventral forebrain organoid induction was performed with dual SMAD and Wnt signalling inhibition, together with additional activation of the Shh pathway. Cerebral organoids and dorsal forebrain model presented the most cell types between days 35 and 60. At 84 days, dorsal forebrain organoids contain astrocytes and potentially oligodendrocytes. Immunofluorescence analysis showed cell type-specific application of non-exclusive markers for multiple cell types. CONCLUSIONS: We provide an easily accessible overview of human brain organoid cultures, which may help those working with brain organoids to define their choice of model, culture time, functional assay, differentiation, and characterisation strategies.

Transmission of Zika virus by dendritic cell subsets in skin and vaginal mucosa.

Zika virus is a member of the Flaviviridae family that has caused recent outbreaks associated with neurological malformations. Transmission of Zika virus occurs primarily via mosquito bite but also via sexual contact. Dendritic cells (DCs) and Langerhans cells (LCs) are important antigen presenting cells in skin and vaginal mucosa and paramount to induce antiviral immunity. To date, little is known about the first cells targeted by Zika virus in these tissues as well as subsequent dissemination of the virus to other target cells. We therefore investigated the role of DCs and LCs in Zika virus infection. Human monocyte derived DCs (moDCs) were isolated from blood and primary immature LCs were obtained from human skin and vaginal explants. Zika virus exposure to moDCs but not skin and vaginal LCs induced Type I Interferon responses. Zika virus efficiently infected moDCs but neither epidermal nor vaginal LCs became infected. Infection of a human full skin model showed that DC-SIGN expressing dermal DCs are preferentially infected over langerin+ LCs. Notably, not only moDCs but also skin and vaginal LCs efficiently transmitted Zika virus to target cells. Transmission by LCs was independent of direct infection of LCs. These data suggest that DCs and LCs are among the first target cells for Zika virus not only in the skin but also the genital tract. The role of vaginal LCs in dissemination of Zika virus from the vaginal mucosa further emphasizes the threat of sexual transmission and supports the investigation of prophylaxes that go beyond mosquito control.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Human Dendritic Cells Transmit Enterovirus A71 via Heparan Sulfates to Target Cells Independent of Viral Replication.

Enterovirus A71 (EV-A71) is a causative agent of life-threatening neurological diseases in young children. EV-A71 is highly infectious but it remains unclear how the virus disseminates from primary entry sites-the mucosa of the respiratory tract or the intestine-to secondary replication sites-skin or brain. Here, we investigated the role of dendritic cells (DCs) in EV-A71 dissemination. DCs reside in the mucosa of the airway and gut, and migrate to lymphoid tissues upon activation and, therefore, could facilitate EV-A71 dissemination to secondary replication sites. Monocyte-derived DCs were not permissive to different genotypes of EV-A71 but, notably, coculture with EV-A71-susceptiblle RD99 cells led to very efficient infection of RD99 cells. Notably, EV-A71 transmission of DCs to RD99 was independent of viral replication as a replication inhibitor did not affect transmission. Soluble heparin blocked EV-A71 transmission by DCs to RD99 cells, in contrast to antibodies against known attachment receptor DC-SIGN. These results strongly suggest that DCs might be a first target for EV-A71 and involved in viral dissemination via heparan sulfates and heparin derivatives might be an effective treatment to attenuate dissemination. IMPORTANCE EV-A71 is an emerging neurotropic virus that is of emerging concern and can result in polio-like illness. The exact mechanism of how EV-A71 results in neurological symptoms are unknown. In particular, the early dissemination of the virus from primary replication sites (airway and intestine) to secondary sites (central nervous system and skin) needs to be elucidated. There is evidence pointing toward a role for dendritic cells (DC) in EV-A71 transmission. Moreover, heparan sulfate (HS) binding mutations are observed in patients with severe diseases. Therefore, we evaluated the potential role of HS on DC in transmission. We find that HS are critical for transmitting EV-A71 by DC to target cells. Our data are consistent with other clinical and in vitro observations highlighting the importance of HS in EV-A71-induced disease.

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.

Enterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells. IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Human milk inhibits some enveloped virus infections, including SARS-CoV-2, in an intestinal model.

Human milk is important for antimicrobial defense in infants and has well demonstrated antiviral activity. We evaluated the protective ability of human milk against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a human fetal intestinal cell culture model. We found that, in this model, human milk blocks SARS-CoV-2 replication, irrespective of the presence of SARS-CoV-2 spike-specific antibodies. Complete inhibition of both enveloped Middle East respiratory syndrome coronavirus and human respiratory syncytial virus infections was also observed, whereas no inhibition of non-enveloped enterovirus A71 infection was seen. Transcriptome analysis after 24 h of the intestinal monolayers treated with human milk showed large transcriptomic changes from human milk treatment, and subsequent analysis suggested that <i>ATP1A1</i> down-regulation by milk might be of importance. Inhibition of ATP1A1 blocked SARS-CoV-2 infection in our intestinal model, whereas no effect on EV-A71 infection was seen. Our data indicate that human milk has potent antiviral activity against particular (enveloped) viruses by potentially blocking the ATP1A1-mediated endocytic process.

Apical-out airway organoids as a platform for studying viral infections and screening for antiviral drugs.

Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air-Liquid Interface (ALI) models, yet the limited accessibility of the organoids' apical side has hindered their applications in studies focusing on host-pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed. These apical-out airway organoids are generated in an Extracellular Matrix (ECM)-free environment from 2D-expanded bronchial epithelial cells and differentiated in suspension to develop uniformly-sized organoid cultures with robust ciliogenesis. Differentiated apical-out airway organoids are susceptible to infection with common respiratory viruses and show varying responses upon treatment with antivirals. In addition to the ease of apical accessibility, these apical-out airway organoids offer an alternative in vitro model to study host-pathogen interactions in higher throughput than the traditional air-liquid interface model.

Detection of intrathecal antibodies to diagnose enterovirus infections of the central nervous system.

BACKGROUND: Enterovirus-D68 (EV-D68) predominantly causes respiratory disease. However, EV-D68 infections also have been associated with central nervous system (CNS) complications, most specifically acute flaccid myelitis (AFM). Diagnosing EV-D68-associated CNS disease is challenging since viral RNA is rarely detected in cerebrospinal fluid (CSF). OBJECTIVE: In order to determine an EV antibody index (AI), we evaluated the value of a commercially available quantitative ELISA to detect EV-specific antibodies in paired CSF and blood. STUDY DESIGN: Nine paired CSF and blood samples were obtained from patients with EV-D68-associated AFM or from patients with a confirmed EV-associated CNS disease. EV-specific antibodies were detected using a quantitative ELISA. A Reiber diagram analysis was performed, by which the AI was calculated. Subsequently, EV ELISA results were compared with an EV-D68 virus neutralization test. RESULTS: ELISA detected EV-specific antibodies in 1 out of the 3 patients with EV-D68-associated AFM and in 3 out of the 6 patients with confirmed EV-associated CNS disease. In these patients, the AI was indicative for intrathecal antibody production against enterovirus. Assay comparison showed that EV-D68 neutralizing antibody detection increased the sensitivity of EV-D68 antibody detection. CONCLUSIONS: A quantitative EV IgG ELISA in combination with Reiber diagram analysis and AI-calculation can be used as a diagnostic tool for EV-associated CNS disease, including EV-D68. An EV-D68 specific ELISA will improve the sensitivity of the tool. With the growing awareness that the detection of non-polio enteroviruses needs to be improved, diagnostic laboratories should consider implementation of EV serology.

Human Brain Organoids as Models for Central Nervous System Viral Infection.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.