Ebola virus (EBOV) disease (EVD) is a hemorrhagic disease caused by EBOV infection. EVD outcomes in pregnant women are similar to non-pregnant women however, EVD is associated with negative fetal outcomes in ∼99% of cases. There is a critical need for a tractable small animal model to study maternal/fetal transmission of EBOV. We utilized interferon α/β receptor knock out mice infected and authentic EBOV or the model virus, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (rVSV/EBOV). Infection with either virus during late pregnancy resulted in placental infection and vertical transmission to the fetus within 2-3 days. Robust levels of maternal and fetal proinflammatory cytokines were evident by day 5 after EBOV infection. Within the placenta, trophoblasts and endothelial cells were viral antigen positive. Elimination of the endosomal receptor NPC1 in junctional zone trophoblasts reduced placental infection and virus transmission to the fetus. These studies establish an infectious model that provides EBOV trafficking and pathogenesis insights during pregnancy. Teaser:This model provides key insights into how viral trafficking and maternal immune responses drive adverse fetal outcomes during gestational Ebola virus infection.

Highly pathogenic viruses in the Filoviridae family are causative agents of filovirus disease (FVD). Ebola virus (EBOV) is one such member and, of all filoviruses, represents the largest threat to global public health. The study of FVD has been hampered by the lack of tools to study filovirus infection outside maximum containment laboratories. Recombinant vesicular stomatitis virus (VSV) lacking its native glycoprotein and expressing a filovirus glycoprotein (VSV-filo GP) has improved our understanding of GP-mediated host-cell interactions as well as adaptive and humoral immune responses in in vitro and in vivo studies. Furthermore, mouse models suitable for these studies are readily available. Here, we describe multiple injection routes for investigating filovirus GP-mediated infection and pathogenesis using VSV-filo GP and interferon α/β receptor-deficient (Ifnar-/-) mice as models. These tools can be safely used outside maximum containment laboratories, are cost effective, and easy to manipulate.

Ebola virus (EBOV), the causative agent of Ebola virus disease, remains one of the World Health Organization's top 10 threats to global health. Infectious EBOV virions can be found on the surface of skin late in infection and may be transmitted to others through skin-to-skin contact. We investigate in vivo EBOV tropism and the kinetics of virus movement to and from the skin. Increasing viral loads were detected over time in the skin of EBOV-infected non-human primates and mice, with antigen detected in dermal stromal and immune cells. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a novel mechanism of virus egress to the epidermal surface. During late infection, proinflammatory responses were elevated in infected visceral organs but minimal in the skin despite significant viral loads. We observed similar viral trafficking and cell tropism in the skin of mice intraperitoneally infected with a low containment EBOV model virus, rVSV/EBOV GP, allowing more detailed mechanistic studies. Sites of virus infection in the skin were patchy, with intense focal areas of infection surrounded by uninfected areas. To investigate virus entry into the body through skin, rVSV/EBOV GP was applied to the surface of gently abraded skin to remove the stratum corneum; epidermal keratinocytes were robustly infected with subsequent systemic viral dissemination observed in some mice. Optimal levels of infection within the skin required expression of the phosphatidylserine receptor, AXL. Collectively, our data demonstrate that skin serves as an important organ targeted by EBOV, facilitating virus entry into and egress from the body. IMPORTANCE Ebola virus (EBOV) remains one of the World Health Organization's top 10 threats to global health, despite the availability of a U.S. Food and Drug Administration-approved vaccine. EBOV spreads through human-to-human contact, yet the role of skin in viral transmission remains unclear. Here, we identify skin as a site of EBOV infection, serving as a potential portal for entry into and egress from the body. In vivo, infectious virions and viral RNA increased in the skin over time, localizing to dermal myeloid and stromal cells and to cells within and surrounding hair follicles, suggesting a novel mechanism for viral shedding. Skin infection was patchy and associated with minimal inflammation, despite significant viral loads. Using a surrogate EBOV model, we demonstrate that systemic infection can occur following topical administration through abraded skin and requires phosphatidylserine receptor, AXL, for optimal infection of skin. These findings redefine the role of skin in EBOV pathogenesis, with implications for barrier-targeted interventions.

Ebola virus (EBOV) causes severe human disease. During late infection, EBOV virions are on the skin's surface; however, the permissive skin cell types and the route of virus translocation to the epidermal surface are unknown. We describe a human skin explant model and demonstrate that EBOV infection of human skin via basal media increases in a time-dependent and dose-dependent manner. In the dermis, cells of myeloid, endothelial, and fibroblast origin were EBOV antigen-positive whereas keratinocytes harbored virus in the epidermis. Infectious virus was detected on the apical epidermal surface within 3 days, indicating that virus propagates and traffics through the explants. Purified human fibroblasts and keratinocytes supported EBOV infection ex vivo and both cell types required the phosphatidylserine receptor, AXL, and the endosomal protein, NPC1, for virus entry. This platform identified susceptible cell types and demonstrated dynamic trafficking of EBOV virions. These findings may explain person-to-person transmission via skin contact.

The filovirus, Ebola virus (EBOV), causes outbreaks of EBOV disease (EVD) throughout equatorial Africa. ERVEBO is a replication-competent recombinant vesicular stomatitis virus-vectored vaccine encoding the EBOV glycoprotein (recombinant vesicular stomatitis virus [rVSV]/EBOV), which is licensed to control EVD outbreaks. EVD outbreaks occur in regions endemic for Plasmodium-caused malaria. Plasmodium infections persist due in part to the parasite's ability to evade sterilizing immunity, which also dampens immune responses to heterologous vaccines. Acute murine Plasmodium infection at the time of rVSV/EBOV vaccination reduced vaccine-mediated protection against mouse-adapted EBOV (ma-EBOV) challenge. Decreased protection was associated with a Plasmodium-induced interferon gamma-mediated decrease of rVSV/EBOV replication in lymph node macrophages, resulting in reduced primary anti-EBOV glycoprotein antibody responses. Higher doses of rVSV/EBOV partially overcame the antibody deficits and elicited protective responses. Evidence of the negative impact of Plasmodium on the efficacy of low-dose rVSV/EBOV vaccine protocols supports the use of high antigen loads in the effective management of EVD outbreaks. IMPORTANCE:We show that a blood-stage murine Plasmodium infection negatively impacts the primary antibody response elicited by low-dose recombinant vesicular stomatitis virus (rVSV)/Ebola virus (EBOV) vaccination and results in reduced protection against a lethal dose of mouse-adapted EBOV. This defect occurs within the draining lymph node due to the elevation of interferon gamma elicited in Plasmodium yoelii (Py)-infected mice. The Py-imposed decrease in vaccine-mediated protection can be overcome with higher doses of rVSV/EBOV. While the strong protection conferred by rVSV/EBOV and significant side effects known to be associated with this vaccine have led to the suggestion that the vaccine dosage be reduced, our studies provide a rationale for maintaining the current higher dose.

Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), remains one of WHO’s top ten threats to global health. Infectious EBOV virions can be found on the surface of skin late during systemic infection and passed from the deceased through skin-to-skin contact. Here, we assess viral load and antigen expression in the skin of EBOV-infected non-human primates (NHP) and mouse adapted-EBOV (ma-EBOV) - infected mice and use the low containment viral model, rVSV/EBOV GP, to mechanistically define skin infection in mice. Viral RNA peaked within the skin proximal to the site of injection in EBOV-infected NHPs on day 6. In contrast, mouse skin sites distal to the site of ma-EBOV injection achieved maximal viral loads by day 3. At late times of infection, viral antigen-positive cells co-localized with markers for endothelial, stromal, and immune cells in the dermis. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a potential mechanism of virus trafficking to the epidermal surface. Despite robust viral infection, distal skin sites of ma-EBOV-infected mice had low expression of proinflammatory stimulated genes. A similar cellular tropism was observed in the skin of mice infected with rVSV/EBOV GP, with discrete focal areas of intense infection. When virus was applied to the surface of gently abraded skin to remove the stratum corneum, epidermal keratinocytes were robustly infected, followed by systemic viral dissemination. To define cell surface receptors critical for virus trafficking to and replication within the skin, mice lacking the phosphatidylserine receptors were infected intraperitoneally with rVSV/EBOV GP. At day 3 of infection, skin distal to the site of infection of TIM-1 knock out (KO) mice had significantly lower levels of infectious virus than the control mice, suggesting that TIM-1 is essential for efficient distribution of virus to the skin. Our findings reveal that EBOV targets specific skin cell populations at late times of viral infection and that the host receptor TIM-1 is required for optimal viral dissemination. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND AND AIMS:Guillain-Barré syndrome (GBS) is a rare disorder, with a global incidence ranging from 1 to 2 individuals per 100,000 people/year. Infections and vaccines have been implicated as causes triggering GBS. The aim of the study was to identify host genes involved in the pathogenesis of GBS when Zika (ZIKV) and Chikungunya viruses (CHIKV) were introduced in Brazil. METHODS:A case-control study of GBS was performed when ZIKV and CHIKV were introduced into a naïve population. GBS was studied during both acute and postacute phases. RNA sequencing was conducted using whole blood. RESULTS:GBS typically manifested a week after rash and fever; acute inflammatory demyelinating polyradiculoneuropathy was more frequent. None of the GBS cases had a poor outcome. Serological assays for ZIKV and CHIKV revealed high titers of immunoglobulin G for both viruses in 9 out of 11 subjects. Metatranscriptomic analyses unveiled an increased abundance of reads attributed to Pseudomonas tolaasii and Toxoplasma gondii in the acute phase. Analysis of differentially expressed host genes during the acute phase revealed altered expression of genes associated with axogenesis, synapse assembly, and presynapse organization. Moreover, genes upregulated during acute GBS were primarily related to inflammation and the inflammasome pathways, including AIM2, NLR family genes and LRR-protein genes, and IL-10. INTERPRETATION:These findings suggest that inflammasome activation via AIM2 could play a role in tissue damage during GBS. Further investigation into the general activation of innate inflammatory responses is warranted to elucidate their potential contribution to the pathology of GBS.

Orthoebolavirus zairense is the species name for Zaire Ebola virus (EBOV) within Filoviridae. This group of viruses can cause severe disease in humans, characterized by hemorrhagic shock, coagulation abnormalities, and severe inflammation. While tissue macrophages are critical targets early during EBOV infection, other cell types support viral replication as disease progresses. At late stages of infection, infectious EBOV is found on the surface of the skin, which may be a critical source of infectious virus transmitted between individuals during outbreaks. Human skin contains a number of cellular targets of EBOV, including keratinocytes. Here, we demonstrate EBOV infection of telomerase-immortalized normal human skin keratinocytes (NHSK-1), as well as EBOVΔVP30 infection of NHSK-1 cells that were stably complemented with EBOV transcription factor VP30. Infection with EBOVΔVP30 did not elicit detectable endogenous interferon responses; however, exogenous pre-treatment of NHSK-1 cells with type I, II, and III interferon (IFN) inhibited EBOVΔVP30 infection and infection of an additional low-containment model of EBOV, rVSV/EBOV GP, in a dose-dependent manner. Analysis of the transcriptome of IFN-treated keratinocytes identified multiple genes unique to each IFN and a subset of ISGs upregulated by all three IFNs. Our results indicate that ISGs induced by IFN pre-treatment of keratinocytes can reduce infection, underlining that ISGs may serve as EBOV-targeting therapeutics.

Post-translational modification of proteins by the addition of sugar chains, or glycans, is a functionally important hallmark of proteins trafficked through the secretory system. These proteins are termed glycoproteins. Glycans are known to be important for initiating signaling through binding of cell surface receptors, facilitating protein folding, and maintaining protein stability. For pathogens, glycans can also mask vulnerable protein regions from neutralizing antibodies. Thus, there is a need to develop methods to decipher the role of specific glycans attached to proteins in order to understand their biological role. Here, we describe established methods for identifying glycosylated residues and understanding their role in protein synthesis and function using viral glycoproteins as a model.

Ebola virus disease (EVD) represents a global health threat. The etiological agents of EVD are six species of Orthoebolaviruses, with Orthoebolavirus zairense (EBOV) having the greatest public health and medical significance. EVD pathogenesis occurs as a result of broad cellular tropism of the virus, robust viral replication and a potent and dysregulated production of cytokines. In vivo, tissue macrophages are some of the earliest cells infected and contribute significantly to virus load and cytokine production. While EBOV is known to infect macrophages and to generate high titer virus in the liver, EBOV infection of liver macrophages, Kupffer cells, has not previously been examined in tissue culture or experimentally manipulated in vivo. Here, we employed primary murine Kupffer cells (KC) and an immortalized murine Kupffer cell line (ImKC) to assess EBOV-eGFP replication in liver macrophages. KCs and ImKCs were highly permissive for EBOV infection and IFN-γ polarization of these cells suppressed their permissiveness to infection. The kinetics of IFN-γ-elicited antiviral responses were examined using a biologically contained model of EBOV infection termed EBOV ΔVP30. The antiviral activity of IFN-γ was transient, but a modest ~3-fold reduction of infection persisted for as long as 6 days post-treatment. To assess the interferon-stimulated gene products (ISGs) responsible for protection, the efficacy of secreted ISGs induced by IFN-γ was evaluated and secreted ISGs failed to block EBOV ΔVP30. Our studies define new cellular tools for the study of EBOV infection that can potentially aid the development of new antiviral therapies. Furthermore, our data underscore the importance of macrophages in EVD pathogenesis and those IFN-γ-elicited ISGs that help to control EBOV infection.

Ebola virus (EBOV) outbreaks are sustained through person-to-person transmission. Anecdotal evidence indicates that virus can be transmitted from EBOV-infected deceased individuals to others during funeral preparations. Consistent with this, viral RNA is detected on the skin at late stages of infection and infectious virus is present in EBOV-infected macaque skin for as long as four days post-mortem. However, the cellular source(s) of infectious virions on the skin have not been widely investigated. The goal of this study was to identify the timing and identity of skin cells infected with EBOV. Human skin explants suspended at the air/liquid interface on transwell inserts were infected with wild-type Ebola virus or a model virus expressing the Ebola GP protein (rVSV/EBOV GP). Explants that were exposed to increasing doses of EBOV in the basolateral media demonstrated dose-dependent levels and timing of infection. Virus replication was evident in the dermis and epidermis as early as day 2 post-infection and the explants supported robust infection as late as day 12. Immunofluorescent staining demonstrated EBOV antigen expression in cytokeratin 5+ keratinocytes. Similar observations were made using our rVSV/EBOV GP model virus; viral titer and viral RNA both increased in a dose and time-dependent fashion along with increasing spread of viral infection in keratinocytes localized to the lower epidermis. Infectious virus was detected on the surface of the explants, suggesting that virus trafficked through the dermis and epidermis to the surface of the skin. Primary human keratinocytes also supported EBOV infection in vitroand infectious virus peaked at days 5-7 pi. This study shows that epidermal keratinocytes can be directly infected by EBOV and are a source of infectious virus indicating that these cells likely play a role in person-to-person transmission through skin contact.

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2′FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.

N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.

STK11 (LKB1) is a tumor suppressor, and loss-of-function mutations contribute to tumorigenesis. Mutations in the STK11 gene (STK11m) are present in ~ 20% of NSCLCs and are associated with poor response to chemotherapy and immune checkpoint inhibition (ICI) resulting in inferior survival outcomes. STK11m tumors are characterized by high oxidative stress/ROS, EMT, enhanced replication stress tolerance, resistance to DNA damage and a highly immunosuppressive tumor microenvironment with limited activation and expansion of anti-tumor CD8 T-cells. AXL, a member of the TAM family of receptor tyrosine kinases, is activated in response to cellular stress such as ROS and hypoxia. AXL expression and activation is associated with EMT and drug resistance, tumor tolerance towards oxidative stress and apoptosis, as well as an immunosuppressed tumor microenvironment. We have previously shown that selective AXL inhibition by bemcentinib potentiated ICI in STK11m NSCLC preclinical models and led to objective clinical response in individuals with STK11m NSCLC receiving bemcentinib and pembrolizumab (NCT03184571). Data from this clinical study, an independent real-world cohort of patients studied at the Haukeland Hospital in Bergen, Norway (REC 45562) and published results1, suggest AXL is expressed in ~ 80% of NSCLCs harboring a STK11m, indicating AXL expression is a characteristic of STK11m NSCLC and confirming that AXL is an attractive target in STK11m NSCLC. Inhibition of AXL in dendritic cells has been shown to increase ICI responses in preclinical models of STK11m NSCLC1. The relative contribution of targeting AXL in STK11m tumor cells vs the tumor microenvironment, and the impact on tumor cells after tumor cell targeting needs further exploration. Transcriptional analysis of STK11m and STK11wt sequences from public datasets, NCT03184571 patients and NSCLC cell lines identified transcriptional signatures consistent with the known roles of STK11 in DNA damage response and immunosuppression. Treatment of STK11m NSCLC cell lines with the AXL inhibitor bemcentinib led to a reduction in the STK11-associated DDR signature and increase in inflammatory signatures demonstrating the impact of AXL targeting on tumor cells. Due to the high unmet medical need in individuals harboring a STK11 mutation, the encouraging efficacy in the NCT03184571 clinical trial and the high incidence of AXL protein expression in STK11m tumors, a global, open-label Phase 1b/2a trial to determine the safety, tolerability and anti-tumor activity of bemcentinib with SOC (pembrolizumab, pemetrexed and carboplatin) in 1L advanced/metastatic non-squamous NSCLC patients with STK11 mutations and no actionable mutations is currently enrolling. The Phase 1b part of the study will evaluate the safety and tolerability of bemcentinib regardless of STK11 status, whereas the Phase 2a part will assess the efficacy in NSCLC patients with STK11 mutations. 1 Li et al., 2022; Cell Rep Med., PMID: 35492873 Citation Format: Magnus Blø, Austin Rayford, Noëlly Madeleine, Fabian Gärtner, Dana Bohan, Natalie Ruggio, Huiyu Li, Luc Girard, Rolf Brekken, John Minna, Marianne Ånerud, Wendy Maury, Claudia Gorcea-Carson, Gro Gausdal, David R. Micklem, Nigel McCracken. AXL as a therapeutic target in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3245.

Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The interhost variability in domain specificity and relative neutralization efficacy of the antibodies is still poorly characterized. To this end, we tested serum and plasma samples collected from 85 coronavirus disease 2019 (COVID-19) convalescent subjects. Samples were analyzed using seven immunoassays that employ different domains, subunits, and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. While the total amount of anti-spike antibodies produced varied among convalescent subjects, we observed an unexpectedly fixed ratio of RBD- to NTD-targeting antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects and was not associated with the overall amount of antispike antibodies produced. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike in early pandemic subjects is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCE Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD and NTD of subunit S1 and against subunit S2. The antibody responses of different infected individuals exhibit different efficacies to inactivate (neutralize) the virus. Here, we show that the observed variation in the neutralizing activity of the antibody responses in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or the potency of their antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ . We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

Adipose tissue is an endocrine organ with strong proinflammatory capacity; however, the role of this tissue in highly pathogenic virus infections has not been extensively examined. We show that mice infected with a mouse-adapted Ebola Virus (EBOV) exhibit increasing levels of viral transcript in visceral and subcutaneous adipose tissue over the course of infection. Human adipocytes were found to be susceptible to EBOV. Endocytosis and macropinocytosis inhibitors effectively blocked infection of adipocytes by a replication competent recombinant VSV virus that expresses EBOV glycoprotein (EBOV-GP/rVSV). While EBOV-GP/rVSV infection of adipocytes caused a robust induction of interferon responsive genes, EBOV infection resulted in modest upregulation of these genes. However, both EBOV-GP/rVSVand EBOV induced comparable and significant induction of the proinflammatory genes CXCL8, IL6, CCL2, and F3 (Tissue Factor). Our results suggest that adipocytes in adipose tissue may contribute to the inflammatory response and coagulopathy that occur during EBOV pathogenesis.