Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity.

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

Betacoronaviruses SARS-CoV-2 and HCoV-OC43 infections in IGROV-1 cell line require aryl hydrocarbon receptor.

The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.

Pteropine orthoreoviruses use cell surface heparan sulphate as an attachment receptor.

Pteropine orthoreoviruses (PRVs) are an emerging group of fusogenic, bat-borne viruses from the Orthoreovirus genus. Since the isolation of PRV from a patient with acute respiratory tract infections in 2006, the zoonotic potential of PRV has been further highlighted following subsequent isolation of PRV species from patients in Malaysia, Hong Kong and Indonesia. However, the entry mechanism of PRV is currently unknown. In this study, we investigated the role of previously identified mammalian orthoreovirus (MRV) receptors, sialic acid and junctional adhesion molecule-1 for PRV infection. However, none of these receptors played a significant role in PRV infection, suggesting PRV uses a distinct entry receptor from MRV. Given its broad tissue tropism, we hypothesized that PRV may use a receptor that is widely expressed in all cell types, heparan sulphate (HS). Enzymatic removal of cell surface HS by heparinase treatment and genetic ablation of HS biosynthesis genes, SLC35B2, exostosin-1, N-deacetylase/N-sulfotransferase I and beta-1,3-glucuronyltransferase 3, significantly reduced infection with multiple genetically distinct PRV species. Replication kinetic of PRV3M in HS knockout cells revealed that HS plays a crucial role in the early phase of PRV infection. Mechanistic studies demonstrated that HS is an essential host-factor for PRV attachment and internalization into cells. To our knowledge, this is the first report on the use of HS as an attachment receptor by PRVs.

Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host-pathogen interactions in the bat airway epithelium.

Bats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host-pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air-liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Single-cell transcriptome analysis of the in vivo response to viral infection in the cave nectar bat Eonycteris spelaea.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

Human Nasal Epithelial Cells (hNECs ) Generated by Air-Liquid Interface (ALI) Culture as a Model System for Studying the Pathogenesis of SARS-CoV-2.

The nasal epithelium lining the human upper airway is the primary portal of entry for several respiratory pathogens, including the recently emerged SARS-CoV-2 virus responsible for the ongoing COVID-19 pandemic. Here, we describe in detail methods for in vitro ALI differentiation of primary cells collected from human donors, to obtain differentiated hNECs. This can serve as a physiologically relevant model to investigate various aspects of host-pathogen responses to SARS-CoV-2 and other emerging respiratory viruses.

Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response.

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.

Immunophenotyping monocytes, macrophages and granulocytes in the Pteropodid bat Eonycteris spelaea.

Bats are asymptomatic reservoir hosts for several highly pathogenic viruses. Understanding this enigmatic relationship between bats and emerging zoonotic viruses requires tools and approaches which enable the comparative study of bat immune cell populations and their functions. We show that bat genomes have a conservation of immune marker genes which delineate phagocyte populations in humans, while lacking key mouse surface markers such as Ly6C and Ly6G. Cross-reactive antibodies against CD44, CD11b, CD14, MHC II, and CD206 were multiplexed to characterize circulating monocytes, granulocytes, bone-marrow derived macrophages (BMDMs) and lung alveolar macrophages (AMs) in the cave nectar bat Eonycteris spelaea. Transcriptional profiling of bat monocytes and BMDMs identified additional markers - including MARCO, CD68, CD163, CD172α, and CD88 - which can be used to further characterize bat myeloid populations. Bat cells often resembled their human counterparts when comparing immune parameters that are divergent between humans and mice, such as the expression patterns of certain immune cell markers. A genome-wide comparison of immune-related genes also revealed a much closer phylogenetic relationship between bats and humans compared to rodents. Taken together, this study provides a set of tools and a comparative framework which will be important for unravelling viral disease tolerance mechanisms in bats.

The proteobacterial species Burkholderia pseudomallei produces ergothioneine, which enhances virulence in mammalian infection.

Bacteria use various endogenous antioxidants for protection against oxidative stress associated with environmental survival or host infection. Although glutathione (GSH) is the most abundant and widely used antioxidant in Proteobacteria, ergothioneine (EGT) is another microbial antioxidant, mainly produced by fungi and Actinobacteria. The Burkholderia genus is found in diverse environmental niches. We observed that gene homologs required for the synthesis of EGT are widely distributed throughout the genus. By generating gene-deletion mutants and monitoring production with isotope-labeled substrates, we show that pathogenic Burkholderia pseudomallei and environmental B. thailandensis are able to synthesize EGT de novo. Unlike most other bacterial EGT synthesis pathways described, Burkholderia spp. use cysteine rather than γ-glutamyl cysteine as the thiol donor. Analysis of recombinant EgtB indicated that it is a proficient sulfoxide synthase, despite divergence in the active site architecture from that of mycobacteria. The absence of GSH, but not EGT, increased bacterial susceptibility to oxidative stresses in vitro. However, deletion of EGT synthesis conferred a reduced fitness to B. pseudomallei, with a delay in organ colonization and time to death during mouse infection. Therefore, despite the lack of an apparent antioxidant role in vitro, EGT is important for optimal bacterial pathogenesis in the mammalian host.-Gamage, A. M., Liao, C., Cheah, I. K., Chen, Y., Lim, D. R. X., Ku, J. W. K., Chee, R. S. L., Gengenbacher, M., Seebeck, F. P., Halliwell, B., Gan, Y.-H. The proteobacterial species Burkholderia pseudomallei produces ergothioneine, which enhances virulence in mammalian infection.

Complement-activated vitronectin enhances the invasion of nonphagocytic cells by bacterial pathogens Burkholderia and Klebsiella.

Burkholderia pseudomallei is a serum-resistant Gram-negative bacterium capable of causing disseminated infections with metastatic complications. However, its interaction with nonphagocytic cells is poorly understood. We observed that exposure of B. pseudomallei and the closely related yet avirulent B. thailandensis to human plasma increased epithelial cell invasion by >20 fold. Enhanced invasion was primarily driven by a plasma factor, which required a functional complement cascade, but surprisingly, was downstream of C3 mediated opsonisation. Receptor blocking studies with RGD-domain containing peptide and αV ß3 blocking antibody identified complement-activated vitronectin as the factor facilitating this invasion. Plasma treatment led to the recruitment of vitronectin onto the bacterial surface, and its conversion into the active conformation. Activation of vitronectin, as well as increased invasion, required the complement pathway and was not observed in C3 or C5 depleted serum. The integrin inhibitor cilengitide, currently in clinical trials as an anti-angiogenesis agent, suppresses plasma-mediated Burkholderia invasion by ~95%, along with a downstream reduction in intracellular bacterial replication. We extend these findings to serum-resistant Klebsiella pneumoniae as well. Thus, the potential use of commercially available integrin inhibitors as anti-infective agents during selective bacterial infections should be explored.

Effect of oral N-acetyl cysteine supplementation in type 2 diabetic patients on intracellular glutathione content and innate immune responses to Burkholderia pseudomallei.

Type 2 diabetic patients have increased susceptibility to melioidosis, an infectious disease caused by Burkholderia pseudomallei. We had previously shown that peripheral blood mononuclear cells (PBMCs) from diabetic patients with poor glycemic control had a defective IL-12 and IFNγ response to B. pseudomallei infection, resulting in poor intracellular bacterial control. The impaired IL-12 response was due to glutathione (GSH) deficiency characterized by a low reduced to oxidized glutathione ratio (GSH ratio) and could be restored by the addition of reduced GSH to the infected cells. Our goal is to determine whether N-acetyl cysteine (NAC, a GSH pro-drug) supplementation in diabetic patients could improve their immune control of B. pseudomallei. Type 2 diabetic patients with poor glycemic control were given oral supplementation of NAC for six weeks at 1200 mg daily. Their PBMCs and subsets of immune cells showed a significant increase in free GSH concentration. However, the GSH ratio, IL-12 and IFNγ production, and intracellular bacterial killing upon ex-vivo infection did not improve. Thus, oral NAC supplementation in diabetic patients is sufficient to increase intracellular GSH content in blood cells. However, modulating the free GSH content is not sufficient to improve infection outcome as it is the GSH ratio that regulates the IL-12 response in monocytes.