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1.
Front Immunol ; 12: 750229, 2021.
Article in English | MEDLINE | ID: covidwho-1506957

ABSTRACT

Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nasal turbinates was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.


Subject(s)
Bronchi/pathology , COVID-19/complications , COVID-19/immunology , Ferrets/immunology , SARS-CoV-2/physiology , Administration, Intranasal , Age Factors , Animals , Asymptomatic Diseases , Disease Models, Animal , Ferrets/virology , Humans , Hyperplasia , Immunity, Cellular , Immunity, Humoral , Injection, Intratympanic , Male , Virus Internalization
2.
J Infect Dis ; 224(8): 1357-1361, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1493824

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Receptors, Coronavirus/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/analysis , Biological Variation, Population , Bronchi/cytology , Bronchi/pathology , Bronchi/virology , COVID-19/virology , Epithelial Cells , Female , Humans , Male , Primary Cell Culture , Receptors, Coronavirus/analysis , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Respiratory Mucosa/virology , Sex Factors , Virus Internalization
3.
Technol Cancer Res Treat ; 20: 15330338211050764, 2021.
Article in English | MEDLINE | ID: covidwho-1477207

ABSTRACT

A pandemic of coronavirus diseases 2019 (COVID-19) outbreak is a major public health emergency that has spread in the fastest speed, and caused the most extensive infection world widely. Transbronchial biopsy (TBB) and computed tomography guided percutaneous needle biopsy (CTPNB) is the most common and significant method for the diagnosis of lung cancer. During the COVID-19 pandemic, the indications of TBB and CTPNB must be managed strictly. Therefore, it is extremely indispensable to perform meticulous and individualized management for lung cancer patients to protect the patients from COVID-19.


Subject(s)
COVID-19/epidemiology , Lung Neoplasms/diagnosis , Biopsy , Bronchi/pathology , Bronchoscopy/methods , COVID-19/complications , COVID-19/diagnosis , Diagnosis, Differential , Disease Susceptibility , Humans , Image-Guided Biopsy/methods , Lung/pathology , Lung Neoplasms/complications , Medical Oncology/methods , Postoperative Period , Prognosis , SARS-CoV-2 , Telemedicine , Tomography, X-Ray Computed
4.
Clin Exp Allergy ; 52(2): 324-333, 2022 02.
Article in English | MEDLINE | ID: covidwho-1437986

ABSTRACT

BACKGROUND: Deaths attributed to Coronavirus Disease 2019 (COVID-19) are mainly due to severe hypoxemic respiratory failure. Although the inflammatory storm has been considered the main pathogenesis of severe COVID-19, hypersensitivity may be another important mechanism involved in severe cases, which have a perfect response to corticosteroids (CS). METHOD: We detected the serum level of anti-SARS-CoV-2-spike S1 protein-specific IgE (SP-IgE) and anti-SARS-CoV-2 nucleocapsid protein-specific IgE (NP-IgE) in COVID-19. Correlation of levels of specific IgE and clinical severity were analysed. Pulmonary function test and bronchial provocation test were conducted in early convalescence of COVID-19. We also obtained histological samples via endoscopy to detect the evidence of mast cell activation. RESULT: The levels of serum SP-IgE and NP-IgE were significantly higher in severe cases, and were correlated with the total lung severity scores (TLSS) and the PaO2 /FiO2 ratio. Nucleocapsid protein could be detected in both airway and intestinal tissues, which was stained positive together with activated mast cells, binded with IgE. Airway hyperresponsiveness (AHR) exists in the early convalescence of COVID-19. After the application of CS in severe COVID-19, SP-IgE and NP-IgE decreased, but maintained at a high level. CONCLUSION: Hypersensitivity may be involved in severe COVID-19.


Subject(s)
Bronchi/immunology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Duodenum/immunology , Hypersensitivity/immunology , Immunoglobulin E/immunology , Mast Cells/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Bronchi/metabolism , Bronchi/pathology , COVID-19/metabolism , COVID-19/pathology , COVID-19/physiopathology , Case-Control Studies , Coronavirus Nucleocapsid Proteins/metabolism , Duodenum/metabolism , Duodenum/pathology , Female , Humans , Hypersensitivity/metabolism , Hypersensitivity/pathology , Hypersensitivity/physiopathology , Lung/physiopathology , Male , Mast Cells/metabolism , Mast Cells/pathology , Middle Aged , Mucous Membrane/immunology , Mucous Membrane/metabolism , Mucous Membrane/pathology , Phosphoproteins/immunology , Phosphoproteins/metabolism , Recovery of Function , Respiratory Hypersensitivity/physiopathology , Retrospective Studies , SARS-CoV-2 , Severity of Illness Index , Spike Glycoprotein, Coronavirus/metabolism , Young Adult
5.
Nat Commun ; 12(1): 5536, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1428813

ABSTRACT

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.


Subject(s)
Endoplasmic Reticulum Stress , SARS-CoV-2/physiology , Virus Replication/physiology , Animals , Autophagy/drug effects , Bronchi/pathology , COVID-19/pathology , COVID-19/virology , Cell Differentiation/drug effects , Cell Extracts , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus 229E, Human/physiology , Down-Regulation/drug effects , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Endoplasmic Reticulum-Associated Degradation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/virology , Heat-Shock Proteins/metabolism , Humans , Macrolides/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Protein Biosynthesis/drug effects , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Thapsigargin/pharmacology , Unfolded Protein Response/drug effects , Vero Cells , Virus Replication/drug effects
6.
Nature ; 583(7818): 834-838, 2020 07.
Article in English | MEDLINE | ID: covidwho-1387423

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/transmission , Coronavirus Infections/virology , Disease Models, Animal , Lung/pathology , Lung/virology , Mesocricetus/virology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Aerosols , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Antigens, Viral/metabolism , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19 , Coronavirus Infections/immunology , Duodenum/virology , Fomites/virology , Housing, Animal , Kidney/virology , Male , Mesocricetus/immunology , Nasal Mucosa/virology , Pandemics , Pneumonia, Viral/immunology , RNA, Viral/analysis , SARS-CoV-2 , Viral Load , Weight Loss
7.
J Cell Mol Med ; 25(14): 7001-7012, 2021 07.
Article in English | MEDLINE | ID: covidwho-1276684

ABSTRACT

The coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in many deaths throughout the world. It is vital to identify the novel prognostic biomarkers and therapeutic targets to assist with the subsequent diagnosis and treatment plan to mitigate the expansion of COVID-19. Since angiotensin-converting enzyme 2 (ACE2)-positive cells are hosts for COVID-19, we focussed on this cell type to explore the underlying mechanisms of COVID-19. In this study, we identified that ACE2-positive cells from the bronchoalveolar lavage fluid (BALF) of patients with COVID-19 belong to bronchial epithelial cells. Comparing with patients of COVID-19 showing severe symptoms, the antigen processing and presentation pathway was increased and 12 typical genes, HLA-DRB5, HLA-DRB1, CD74, HLA-DRA, HLA-DPA1, HLA-DQA1, HSP90AA1, HSP90AB1, HLA-DPB1, HLA-DQB1, HLA-DQA2, and HLA-DMA, particularly HLA-DPB1, were obviously up-regulated in ACE2-positive bronchial epithelial cells of patients with mild disease. We further discovered SDCBP was positively correlated with above 12 genes particularly with HLA-DPB1 in ACE2-positive bronchial epithelial cells of COVID-19 patients. Moreover, SDCBP may increase the immune infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells in different lung carcinoma. Moreover, we found the expression of SDCBP was positively correlated with the expression of antigen processing and presentation genes in post-mortem lung biopsies tissues, which is consistent with previous discoveries. These results suggest that SDCBP has good potential to be further developed as a novel diagnostic and therapeutic target in the treatment of COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Bronchi/pathology , COVID-19/pathology , Epithelial Cells/metabolism , RNA-Seq , Severity of Illness Index , Single-Cell Analysis , Syntenins/metabolism , Antigen Presentation/genetics , Bronchoalveolar Lavage Fluid , COVID-19/genetics , COVID-19/metabolism , Epithelial Cells/pathology , Gene Expression Profiling , Humans , Postmortem Changes , SARS-CoV-2/physiology , Up-Regulation/genetics
8.
STAR Protoc ; 2(3): 100663, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1275773

ABSTRACT

Patients with chronic lung disease are vulnerable to getting severe diseases associated with SARS-CoV-2 infection. Here, we describe protocols for subculturing and differentiating primary normal human bronchial epithelial (NHBE) cells of patients with chronic obstructive lung disease. The differentiation of NHBE cells in air-liquid interface mimics an in vivo airway and provides an in vitro model for studying SARS-CoV-2 infection. We also describe a protocol for detecting proteins in the sectioned epithelium for detailing SARS-CoV-2 infection-induced pathobiology with a vertical view.


Subject(s)
Bronchi/metabolism , COVID-19/complications , Coronavirus Nucleocapsid Proteins/analysis , Epithelium/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , SARS-CoV-2/isolation & purification , Bronchi/pathology , Bronchi/virology , COVID-19/metabolism , COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , Epithelium/pathology , Epithelium/virology , Humans , Immunohistochemistry , Paraffin Embedding , Phosphoproteins/analysis , Phosphoproteins/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/virology , Virus Replication
9.
J Allergy Clin Immunol ; 147(6): 2083-2097.e6, 2021 06.
Article in English | MEDLINE | ID: covidwho-1272498

ABSTRACT

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.


Subject(s)
Bronchi/immunology , COVID-19/immunology , Complement Activation , Epithelial Cells/immunology , Receptor, Anaphylatoxin C5a/immunology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Bronchi/pathology , Bronchi/virology , COVID-19/pathology , COVID-19/virology , Cell Line , Complement C3/immunology , Cytokines/immunology , Epithelial Cells/pathology , Epithelial Cells/virology , Humans , Inflammation/immunology , Inflammation/pathology , Respiratory Mucosa/pathology , Respiratory Mucosa/virology
10.
Int J Mol Sci ; 22(10)2021 May 20.
Article in English | MEDLINE | ID: covidwho-1244036

ABSTRACT

Genome-wide association studies (GWAS) found locus 3p21.31 associated with severe COVID-19. CCR5 resides at the same locus and, given its known biological role in other infection diseases, we investigated if common noncoding and rare coding variants, affecting CCR5, can predispose to severe COVID-19. We combined single nucleotide polymorphisms (SNPs) that met the suggestive significance level (P ≤ 1 × 10-5) at the 3p21.31 locus in public GWAS datasets (6406 COVID-19 hospitalized patients and 902,088 controls) with gene expression data from 208 lung tissues, Hi-C, and Chip-seq data. Through whole exome sequencing (WES), we explored rare coding variants in 147 severe COVID-19 patients. We identified three SNPs (rs9845542, rs12639314, and rs35951367) associated with severe COVID-19 whose risk alleles correlated with low CCR5 expression in lung tissues. The rs35951367 resided in a CTFC binding site that interacts with CCR5 gene in lung tissues and was confirmed to be associated with severe COVID-19 in two independent datasets. We also identified a rare coding variant (rs34418657) associated with the risk of developing severe COVID-19. Our results suggest a biological role of CCR5 in the progression of COVID-19 as common and rare genetic variants can increase the risk of developing severe COVID-19 by affecting the functions of CCR5.


Subject(s)
COVID-19/genetics , COVID-19/metabolism , Genetic Predisposition to Disease , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Alleles , Bronchi/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19/physiopathology , Chromosomes, Human/genetics , Cohort Studies , Computational Biology , Databases, Genetic , Genome-Wide Association Study , Genotype , Humans , Lung/metabolism , Lung/pathology , Lung/virology , Polymorphism, Single Nucleotide , Whole Exome Sequencing
11.
Am J Pathol ; 191(8): 1374-1384, 2021 08.
Article in English | MEDLINE | ID: covidwho-1240148

ABSTRACT

Patients with coronavirus disease 2019 (COVID-19) who are critically ill develop vascular complications characterized by thrombosis of small, medium, and large vessels. Dysfunction of the vascular endothelium due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated in the pathogenesis of the COVID-19 vasculopathy. Although initial reports suggested that endothelial injury was caused directly by the virus, recent studies indicate that endothelial cells do not express angiotensin-converting enzyme 2, the receptor that SARS-CoV-2 uses to gain entry into cells, or express it at low levels and are resistant to the infection. These new findings, together with the observation that COVID-19 triggers a cytokine storm capable of injuring the endothelium and disrupting its antithrombogenic properties, favor an indirect mechanism of endothelial injury mediated locally by an augmented inflammatory reaction to infected nonendothelial cells, such as the bronchial and alveolar epithelium, and systemically by the excessive immune response to infection. Herein we review the vascular pathology of COVID-19 and critically discuss the potential mechanisms of endothelial injury in this disease.


Subject(s)
COVID-19/metabolism , Cytokine Release Syndrome/metabolism , Endothelium, Vascular/injuries , Endothelium, Vascular/metabolism , SARS-CoV-2/metabolism , Thrombosis/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Bronchi/pathology , COVID-19/complications , COVID-19/pathology , COVID-19/therapy , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/therapy , Endothelium, Vascular/pathology , Humans , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Thrombosis/etiology , Thrombosis/pathology , Thrombosis/therapy
12.
PLoS Biol ; 19(3): e3001006, 2021 03.
Article in English | MEDLINE | ID: covidwho-1148237

ABSTRACT

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.


Subject(s)
SARS-CoV-2/physiology , Virus Replication/physiology , Amino Acid Substitution , Animals , Base Sequence , Bronchi/pathology , COVID-19/diagnosis , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Epithelial Cells/pathology , Epithelial Cells/virology , Furin/metabolism , Host-Pathogen Interactions , Humans , SARS-CoV-2/isolation & purification , Vero Cells
13.
PLoS Biol ; 19(3): e3001143, 2021 03.
Article in English | MEDLINE | ID: covidwho-1138557

ABSTRACT

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.


Subject(s)
Bronchi/pathology , COVID-19/diagnosis , Gene Expression , SARS-CoV-2/isolation & purification , Single-Cell Analysis/methods , Adult , Bronchi/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Cells, Cultured , Epithelium/pathology , Epithelium/virology , Humans , Immunity, Innate , Longitudinal Studies , SARS-CoV-2/genetics , Transcriptome , Viral Tropism
14.
J Cell Physiol ; 236(9): 6597-6606, 2021 09.
Article in English | MEDLINE | ID: covidwho-1098899

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/drug therapy , HMGB1 Protein/genetics , Proto-Oncogene Proteins c-akt/genetics , A549 Cells , Bronchi/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19/genetics , COVID-19/pathology , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation/drug effects , Humans , Lung/drug effects , Lung/pathology , RNA, Viral/genetics , Ribonucleosides/administration & dosage , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
15.
Gastroenterology ; 160(5): 1647-1661, 2021 04.
Article in English | MEDLINE | ID: covidwho-1065985

ABSTRACT

BACKGROUND & AIMS: Gastrointestinal (GI) manifestations have been increasingly reported in patients with coronavirus disease 2019 (COVID-19). However, the roles of the GI tract in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We investigated how the GI tract is involved in SARS-CoV-2 infection to elucidate the pathogenesis of COVID-19. METHODS: Our previously established nonhuman primate (NHP) model of COVID-19 was modified in this study to test our hypothesis. Rhesus monkeys were infected with an intragastric or intranasal challenge with SARS-CoV-2. Clinical signs were recorded after infection. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction. Host responses to SARS-CoV-2 infection were evaluated by examining inflammatory cytokines, macrophages, histopathology, and mucin barrier integrity. RESULTS: Intranasal inoculation with SARS-CoV-2 led to infections and pathologic changes not only in respiratory tissues but also in digestive tissues. Expectedly, intragastric inoculation with SARS-CoV-2 resulted in the productive infection of digestive tissues and inflammation in both the lung and digestive tissues. Inflammatory cytokines were induced by both types of inoculation with SARS-CoV-2, consistent with the increased expression of CD68. Immunohistochemistry and Alcian blue/periodic acid-Schiff staining showed decreased Ki67, increased cleaved caspase 3, and decreased numbers of mucin-containing goblet cells, suggesting that the inflammation induced by these 2 types of inoculation with SARS-CoV-2 impaired the GI barrier and caused severe infections. CONCLUSIONS: Both intranasal and intragastric inoculation with SARS-CoV-2 caused pneumonia and GI dysfunction in our rhesus monkey model. Inflammatory cytokines are possible connections for the pathogenesis of SARS-CoV-2 between the respiratory and digestive systems.


Subject(s)
COVID-19/transmission , Gastroenteritis/pathology , Gastrointestinal Tract/pathology , Lung/pathology , Animals , Bronchi/metabolism , Bronchi/pathology , COVID-19/immunology , COVID-19/metabolism , COVID-19/pathology , COVID-19 Nucleic Acid Testing , Caspase 3/metabolism , Cytokines/immunology , Disease Models, Animal , Gastric Mucosa , Gastroenteritis/metabolism , Gastroenteritis/virology , Gastrointestinal Tract/immunology , Gastrointestinal Tract/metabolism , Goblet Cells/pathology , Intestine, Small/metabolism , Intestine, Small/pathology , Ki-67 Antigen/metabolism , Lung/diagnostic imaging , Lung/immunology , Lung/metabolism , Macaca mulatta , Nasal Mucosa , RNA, Viral/isolation & purification , Random Allocation , Rectum/metabolism , Rectum/pathology , SARS-CoV-2 , Trachea/metabolism , Trachea/pathology
16.
Virchows Arch ; 479(4): 827-833, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1012211

ABSTRACT

We present results from clinical, radiologic, gas exchange, lung mechanics, and fibre-optic bronchoscopy-guided transbronchial biopsies in a case of acute respiratory failure due to SARS-CoV-2 (Covid-19). This report highlights the pulmonary, immunological, and inflammatory changes found during acute diffuse alveolar damage and the later organizing phase. An early diffuse alveolar damage pattern with predominant epithelial involvement with active recruitment of T cells and monocytes was observed followed by a late organizing pattern with pneumocyte hyperplasia, inflammatory infiltration, prominent endotheliitis, and secondary germinal centers. The patient's deterioration paralleling the late immuno-pathological findings based the decision to administer intravenous corticosteroids, resulting in clinical, gasometric, and radiologic improvement. We believe that real-time clinicopathological correlation, along with the description of the immunological processes at play, will contribute to the full clinical picture of Covid-19 and might lead to a more rational approach in the precise timing of anti-inflammatory, anti-cytokine, or steroid therapies.


Subject(s)
Bronchi/pathology , COVID-19/drug therapy , Steroids/therapeutic use , Aged , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Biopsy/methods , Bronchi/virology , COVID-19/pathology , COVID-19/virology , Humans , Lung/pathology , Male , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/virology , Respiratory Insufficiency/drug therapy , Respiratory Insufficiency/pathology , Respiratory Insufficiency/virology , SARS-CoV-2/isolation & purification
17.
Eur Radiol ; 31(1): 121-130, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-691583

ABSTRACT

OBJECTIVES: CT findings of COVID-19 look similar to other atypical and viral (non-COVID-19) pneumonia diseases. This study proposes a clinical computer-aided diagnosis (CAD) system using CT features to automatically discriminate COVID-19 from non-COVID-19 pneumonia patients. METHODS: Overall, 612 patients (306 COVID-19 and 306 non-COVID-19 pneumonia) were recruited. Twenty radiological features were extracted from CT images to evaluate the pattern, location, and distribution of lesions of patients in both groups. All significant CT features were fed in five classifiers namely decision tree, K-nearest neighbor, naïve Bayes, support vector machine, and ensemble to evaluate the best performing CAD system in classifying COVID-19 and non-COVID-19 cases. RESULTS: Location and distribution pattern of involvement, number of the lesion, ground-glass opacity (GGO) and crazy-paving, consolidation, reticular, bronchial wall thickening, nodule, air bronchogram, cavity, pleural effusion, pleural thickening, and lymphadenopathy are the significant features to classify COVID-19 from non-COVID-19 groups. Our proposed CAD system obtained the sensitivity, specificity, and accuracy of 0.965, 93.54%, 90.32%, and 91.94%, respectively, using ensemble (COVIDiag) classifier. CONCLUSIONS: This study proposed a COVIDiag model obtained promising results using CT radiological routine features. It can be considered an adjunct tool by the radiologists during the current COVID-19 pandemic to make an accurate diagnosis. KEY POINTS: • Location and distribution of involvement, number of lesions, GGO and crazy-paving, consolidation, reticular, bronchial wall thickening, nodule, air bronchogram, cavity, pleural effusion, pleural thickening, and lymphadenopathy are the significant features between COVID-19 from non-COVID-19 groups. • The proposed CAD system, COVIDiag, could diagnose COVID-19 pneumonia cases with an AUC of 0.965 (sensitivity = 93.54%; specificity = 90.32%; and accuracy = 91.94%). • The AUC, sensitivity, specificity, and accuracy obtained by radiologist diagnosis are 0.879, 87.10%, 88.71%, and 87.90%, respectively.


Subject(s)
COVID-19/diagnostic imaging , Lung/diagnostic imaging , Tomography, X-Ray Computed , Adult , Aged , Bayes Theorem , Bronchi/diagnostic imaging , Bronchi/pathology , COVID-19/pathology , Diagnosis, Differential , Female , Humans , Lung/pathology , Lymphadenopathy/diagnostic imaging , Lymphadenopathy/pathology , Male , Middle Aged , Pandemics , Pleural Effusion/diagnostic imaging , Retrospective Studies , SARS-CoV-2
18.
Exp Cell Res ; 395(2): 112204, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-680000

ABSTRACT

BACKGROUND: SARS-CoV2, the agent responsible for the current pandemic, is also causing respiratory distress syndrome (RDS), hyperinflammation and high mortality. It is critical to dissect the pathogenetic mechanisms in order to reach a targeted therapeutic approach. METHODS: In the present investigation, we evaluated the effects of SARS-CoV2 on human bronchial epithelial cells (HBEC). We used RNA-seq datasets available online for identifying SARS-CoV2 potential genes target on human bronchial epithelial cells. RNA expression levels and potential cellular gene pathways have been analyzed. In order to identify possible common strategies among the main pandemic viruses, such as SARS-CoV2, SARS-CoV1, MERS-CoV, and H1N1, we carried out a hypergeometric test of the main genes transcribed in the cells of the respiratory tract exposed to these viruses. RESULTS: The analysis showed that two mechanisms are highly regulated in HBEC: the innate immunity recruitment and the disassembly of cilia and cytoskeletal structure. The granulocyte colony-stimulating factor (CSF3) and dynein heavy chain 7, axonemal (DNAH7) represented respectively the most upregulated and downregulated genes belonging to the two mechanisms highlighted above. Furthermore, the carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7) that codifies for a surface protein is highly specific of SARS-CoV2 and not for SARS-CoV1, MERS-CoV, and H1N1, suggesting a potential role in viral entry. In order to identify potential new drugs, using a machine learning approach, we highlighted Flunisolide, Thalidomide, Lenalidomide, Desoximetasone, xylazine, and salmeterol as potential drugs against SARS-CoV2 infection. CONCLUSIONS: Overall, lung involvement and RDS could be generated by the activation and down regulation of diverse gene pathway involving respiratory cilia and muscle contraction, apoptotic phenomena, matrix destructuration, collagen deposition, neutrophil and macrophages recruitment.


Subject(s)
Bronchi/metabolism , Coronavirus Infections/genetics , Gene Regulatory Networks , Pneumonia, Viral/genetics , Respiratory Mucosa/metabolism , Transcriptome , Bronchi/pathology , COVID-19 , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/metabolism , Coronavirus Infections/metabolism , Drug Discovery/methods , Dyneins/genetics , Dyneins/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Immunity, Innate , Machine Learning , Pandemics , Pneumonia, Viral/metabolism , Up-Regulation
19.
J Allergy Clin Immunol ; 146(2): 315-324.e7, 2020 08.
Article in English | MEDLINE | ID: covidwho-592253

ABSTRACT

BACKGROUND: More than 300 million people carry a diagnosis of asthma, with data to suggest that they are at a higher risk for infection or adverse outcomes from severe acute respiratory syndrome coronavirus 2. Asthma is remarkably heterogeneous, and it is currently unclear how patient-intrinsic factors may relate to coronavirus disease 2019. OBJECTIVE: We sought to identify and characterize subsets of patients with asthma at increased risk for severe acute respiratory syndrome coronavirus 2 infection. METHODS: Participants from 2 large asthma cohorts were stratified using clinically relevant parameters to identify factors related to angiotensin-converting enzyme-2 (ACE2) expression within bronchial epithelium. ACE-2-correlated gene signatures were used to interrogate publicly available databases to identify upstream signaling events and novel therapeutic targets. RESULTS: Stratifying by type 2 inflammatory biomarkers, we identified subjects who demonstrated low peripheral blood eosinophils accompanied by increased expression of the severe acute respiratory syndrome coronavirus 2 receptor ACE2 in bronchial epithelium. Genes highly correlated with ACE2 overlapped with type 1 and 2 IFN signatures, normally induced by viral infections. T-cell recruitment and activation within bronchoalveolar lavage cells of ACE2-high subjects was reciprocally increased. These patients demonstrated characteristics corresponding to risk factors for severe coronavirus disease 2019, including male sex, history of hypertension, low peripheral blood, and elevated bronchoalveolar lavage lymphocytes. CONCLUSIONS: ACE2 expression is linked to upregulation of viral response genes in a subset of type 2-low patients with asthma with characteristics resembling known risk factors for severe coronavirus disease 2019. Therapies targeting the IFN family and T-cell-activating factors may therefore be of benefit in a subset of patients.


Subject(s)
Asthma/epidemiology , Asthma/genetics , Coronavirus Infections/epidemiology , Pandemics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/epidemiology , Receptors, Virus/genetics , Adolescent , Adult , Angiotensin-Converting Enzyme 2 , Asthma/classification , Asthma/immunology , Betacoronavirus/genetics , Betacoronavirus/immunology , Biomarkers/metabolism , Bronchi/immunology , Bronchi/pathology , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19 , Cohort Studies , Coronavirus Infections/virology , Eosinophils/immunology , Eosinophils/pathology , Female , Gene Expression Profiling , Humans , Interferon Type I/genetics , Interferon Type I/immunology , Interferon-gamma/genetics , Interferon-gamma/immunology , Male , Middle Aged , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/virology , Protein Interaction Mapping , Receptors, Virus/immunology , Risk Factors , SARS-CoV-2 , Severity of Illness Index , T-Lymphocytes/classification , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Transcriptome , United States/epidemiology
20.
J Renin Angiotensin Aldosterone Syst ; 21(2): 1470320320928872, 2020.
Article in English | MEDLINE | ID: covidwho-543313

ABSTRACT

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus family member that triggers a respiratory disease similar to severe acute respiratory syndrome coronavirus (SARS-CoV). SARS-CoV and SARS-CoV-2 are very similar to each other in many respects, such as structure, genetics, and pathobiology. We hypothesized that coronaviruses could affect pulmonary tissues via integration with the critical immune genes after their interaction with renin-angiotensin system (RAS) elements. The aim of the present bioinformatics study was to assess expression changes of the RAS and non-RAS genes, particularly immune response genes, in the lung epithelial cells after infection with SARS-CoV. METHODS: Linear regression, hierarchical clustering, pathway analysis, and network analysis were performed using the E-GEOD-17400 data set. RESULTS: The whole-genome expression data of the lung epithelial cells infected with SARS-CoV for 12, 24, and 48 hours were analyzed, and a total of 15 RAS family and 29 immune genes were found to be highly correlated with the exposure time to the virus in the studied groups. CONCLUSION: RAS genes are important at the initiation of the infections caused by coronavirus family members and may have a strong relationship with the exchange of immune genes in due course following the infection.


Subject(s)
Betacoronavirus/physiology , Bronchi/pathology , Coronavirus Infections/genetics , Epithelial Cells/metabolism , Epithelial Cells/virology , Inflammation/genetics , Pneumonia, Viral/genetics , Renin-Angiotensin System/genetics , COVID-19 , Cluster Analysis , Gene Expression Regulation , Gene Regulatory Networks , Genome, Human , Humans , Inflammation/pathology , Linear Models , Pandemics , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2
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