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1.
J Am Soc Nephrol ; 32(1): 99-114, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496673

ABSTRACT

BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.


Subject(s)
Complement C3/genetics , Disease Models, Animal , Glomerulonephritis, Membranoproliferative/genetics , Kidney Diseases/genetics , Animals , Complement C3/metabolism , Complement Pathway, Alternative/genetics , Exons , Gene Expression Regulation , Glomerulonephritis, Membranoproliferative/metabolism , Humans , Kidney Diseases/metabolism , Liver/metabolism , Male , Mice , Mice, Knockout , Microscopy, Fluorescence , Phenotype , Polymorphism, Single Nucleotide , Renal Insufficiency/genetics , Renal Insufficiency/metabolism
2.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1294968

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
3.
Invest Ophthalmol Vis Sci ; 62(7): 25, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1280514

ABSTRACT

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Enzyme Inhibitors/pharmacology , Epithelium, Corneal/enzymology , Gene Expression Regulation, Enzymologic/physiology , Keratitis/enzymology , Resveratrol/pharmacology , SARS-CoV-2/physiology , Adult , Angiotensin-Converting Enzyme 2/metabolism , Animals , Blotting, Western , Cells, Cultured , Epithelium, Corneal/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Inflammation/drug therapy , Inflammation/enzymology , Interleukin-1beta/pharmacology , Keratitis/drug therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Fluorescence , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation
4.
Int J Mol Sci ; 22(12)2021 Jun 18.
Article in English | MEDLINE | ID: covidwho-1273462

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.


Subject(s)
Microscopy, Fluorescence , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Endocytosis , Fluorescent Dyes/chemistry , Genes, Reporter , Humans , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization
6.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1119222

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
8.
Cornea ; 39(12): 1556-1562, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1109355

ABSTRACT

PURPOSE: To confirm the ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by evaluating the expression of viral entry factors in human ocular tissues using immunohistochemistry. METHODS: Fresh donor corneas and primary explant cultures of corneal, limbal, and conjunctival epithelial cells were evaluated for the expression of viral entry factors. Using immunohistochemistry, the samples were tested for the expression of angiotension-converting enzyme 2 (ACE2), dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), DC-SIGN-related protein (DC-SIGNR), and transmembrane serine protease 2 (TMPRSS2). RESULTS: In total, 5 donor corneas were evaluated for the expression of viral entry factors. In all specimens, both ACE2 and TMPRSS2 were expressed throughout the surface epithelium (corneal, limbal, and conjunctival) and corneal endothelium. In corneal stromal cells, ACE2 was sporadically expressed, whereas TMPRSS2 was absent. DC-SIGN/DC-SIGNR expression varied between donor specimens. Four specimens expressed DC-SIGN/DC-SIGNR in a similar distribution to ACE2, but 1 specimen from a young donor showed no expression of DC-SIGN/DC-SIGNR. ACE2, TMPRSS2, and DC-SIGN/DC-SIGNR were all expressed in the cultured corneal, limbal, and conjunctival epithelial cells. CONCLUSIONS: Both corneal and conjunctival epithelia express ACE2, DC-SIGN/DC-SIGNR, and TMPRSS2, suggesting that the ocular surface is a potential route for the transmission of SARS-CoV-2. The risk of viral transmission with corneal transplantation cannot be ruled out, given the presence of ACE2 in corneal epithelium and endothelium. Cultured corneal, limbal, and conjunctival epithelial cells mimic the expression of viral entry factors in fresh donor tissue and may be useful for future in vitro SARS-CoV-2 infection studies.


Subject(s)
Betacoronavirus/physiology , Cell Adhesion Molecules/metabolism , Conjunctiva/metabolism , Epithelium, Corneal/metabolism , Lectins, C-Type/metabolism , Peptidyl-Dipeptidase A/metabolism , Receptors, Cell Surface/metabolism , Serine Endopeptidases/metabolism , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2 , COVID-19 , Cells, Cultured , Conjunctiva/cytology , Coronavirus Infections/immunology , Epithelial Cells/metabolism , Female , Fluorescent Antibody Technique, Indirect , Humans , Limbus Corneae/cytology , Male , Microscopy, Fluorescence , Middle Aged , Pandemics , Pneumonia, Viral/immunology , SARS-CoV-2 , Tissue Donors , Viral Tropism/physiology , Virus Internalization , Young Adult
9.
ACS Appl Mater Interfaces ; 13(8): 10321-10327, 2021 Mar 03.
Article in English | MEDLINE | ID: covidwho-1087402

ABSTRACT

Early diagnosis of SARS-CoV-2 infection is critical for facilitating proper containment procedures, and a rapid, sensitive antigen assay is a critical step in curbing the pandemic. In this work, we report the use of a high-purity semiconducting (sc) single-walled carbon nanotube (SWCNT)-based field-effect transistor (FET) decorated with specific binding chemistry to assess the presence of SARS-CoV-2 antigens in clinical nasopharyngeal samples. Our SWCNT FET sensors, with functionalization of the anti-SARS-CoV-2 spike protein antibody (SAb) and anti-nucleocapsid protein antibody, detected the S antigen (SAg) and N antigen (NAg), reaching a limit of detection of 0.55 fg/mL for SAg and 0.016 fg/mL for NAg in calibration samples. SAb-functionalized FET sensors also exhibited good sensing performance in discriminating positive and negative clinical samples, indicating a proof of principle for use as a rapid COVID-19 antigen diagnostic tool with high analytical sensitivity and specificity at low cost.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques , COVID-19 Testing/instrumentation , Nanotubes, Carbon/chemistry , Semiconductors , Transistors, Electronic , COVID-19 Testing/methods , Calibration , Electrodes , Gold , Humans , Limit of Detection , Materials Testing , Microscopy, Atomic Force , Microscopy, Fluorescence , Nanotechnology , SARS-CoV-2 , Sensitivity and Specificity , Spectrophotometry, Ultraviolet , Spectroscopy, Near-Infrared , Spectrum Analysis, Raman , Spike Glycoprotein, Coronavirus/analysis
10.
ACS Appl Bio Mater ; 4(2): 1307-1318, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1069089

ABSTRACT

Recent evidence suggests that proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), play a pivotal role in the development of inflammatory-related pathologies (covid-19, depressive disorders, sepsis, cancer, etc.,). More importantly, the development of TNF-α biosensors applied to biological fluids (e.g. sweat) could offer non-invasive solutions for the continuous monitoring of these disorders, in particular, polydimethylsiloxane (PDMS)-based biosensors. We have therefore investigated the biofunctionalization of PDMS surfaces using a silanization reaction with 3-aminopropyltriethoxysilane, for the development of a human TNF-α biosensor. The silanization conditions for 50 µm PDMS surfaces were extensively studied by using water contact angle measurements, electron dispersive X-ray and Fourier transform infrared spectroscopies, and fluorescamine detection. Evaluation of the wettability of the silanized surfaces and the Si/C ratio pointed out to the optimal silanization conditions supporting the formation of a stable and reproducible aminosilane layer, necessary for further bioconjugation. An ELISA-type immunoassay was then successfully performed for the detection and quantification of human TNF-α through fluorescent microscopy, reaching a limit of detection of 0.55 µg/mL (31.6 nM). Finally, this study reports for the first time a promising method for the development of PDMS-based biosensors for the detection of TNF-α, using a quick, stable, and simple biofunctionalization process.


Subject(s)
Dimethylpolysiloxanes/chemistry , Immunoassay/methods , Tumor Necrosis Factor-alpha/analysis , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Carbon/chemistry , Humans , Immunoassay/instrumentation , Limit of Detection , Microfluidics , Microscopy, Fluorescence , SARS-CoV-2/isolation & purification , Silicon/chemistry , Tumor Necrosis Factor-alpha/immunology , Wettability
11.
Sci Rep ; 11(1): 1822, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-1065937

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID-19 global pandemic has infected over 25 million people worldwide and resulted in the death of millions. The COVID-19 pandemic has also resulted in a shortage of personal protective equipment (PPE) in many regions around the world, particularly in middle- and low-income countries. The shortages of PPE, such as N95 respirators, is something that will persist until an effective vaccine is made available. Thus, devices that while being easy to operate can also be rapidly deployed in health centers, and long-term residences without the need for major structural overhaul are instrumental to sustainably use N95 respirators. In this report, we present the design and validation of a decontamination device that combines UV-C & B irradiation with mild-temperature treatment. The device can decontaminate up to 20 masks in a cycle of < 30 min. The decontamination process did not damage or reduce the filtering capacity of the masks. Further, the efficacy of the device to eliminate microbes and viruses from the masks was also evaluated. The photothermal treatment of our device was capable of eradicating > 99.9999% of the bacteria and > 99.99% of the virus tested.


Subject(s)
Bacteria/radiation effects , Decontamination/methods , Ultraviolet Rays , Viruses/radiation effects , COVID-19/pathology , COVID-19/virology , Equipment Reuse , HEK293 Cells , Humans , Microscopy, Fluorescence , N95 Respirators/virology , SARS-CoV-2/isolation & purification , SARS-CoV-2/radiation effects , Temperature , Viruses/metabolism
12.
EBioMedicine ; 63: 103182, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1014448

ABSTRACT

BACKGROUND: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. METHODS: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. FINDINGS: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. INTERPRETATION: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. FUNDING: No external funding was necessary for this study.


Subject(s)
Capillaries/pathology , Coronary Vessels/pathology , SARS-CoV-2/metabolism , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Capillaries/metabolism , Coronary Vessels/metabolism , Female , Humans , Inflammation/pathology , Male , Microscopy, Fluorescence , Middle Aged , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
13.
Nat Commun ; 11(1): 6041, 2020 11 27.
Article in English | MEDLINE | ID: covidwho-947535

ABSTRACT

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/epidemiology , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HeLa Cells , Humans , Insecta , Intravital Microscopy , Microscopy, Fluorescence , Molecular Dynamics Simulation , Pandemics/prevention & control , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Transcription/drug effects , Viral Transcription/physiology , Virus Replication/drug effects , Virus Replication/genetics
14.
J Virol ; 94(22)2020 10 27.
Article in English | MEDLINE | ID: covidwho-901265

ABSTRACT

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro 3CLpro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CLpro Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CLpro This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread.IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/chemistry , Coronavirus Infections/virology , Drug Discovery , High-Throughput Screening Assays/methods , Pneumonia, Viral/virology , Protease Inhibitors/pharmacology , Animals , COVID-19 , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus Infections/drug therapy , Cysteine Endopeptidases , Humans , Microscopy, Fluorescence/methods , Pandemics , Pneumonia, Viral/drug therapy , SARS-CoV-2 , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors
15.
Antiviral Res ; 184: 104955, 2020 12.
Article in English | MEDLINE | ID: covidwho-871719

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral activity and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , COVID-19/virology , Myocytes, Cardiac/virology , Pluripotent Stem Cells/cytology , SARS-CoV-2/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Amides/pharmacology , Animals , Antimalarials/pharmacology , COVID-19/complications , COVID-19/drug therapy , Chlorocebus aethiops , Chloroquine/pharmacology , Electrocardiography , Flow Cytometry , Heart Diseases/complications , Humans , Hydroxychloroquine/pharmacology , Microscopy, Fluorescence , Myocytes, Cardiac/drug effects , Pluripotent Stem Cells/virology , Pyrazines/pharmacology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Vero Cells , Viral Plaque Assay
16.
Microsc Res Tech ; 83(12): 1623-1638, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-695947

ABSTRACT

Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoVs), causative of major outbreaks in the past two decades, has claimed many lives all over the world. The virus effectively spreads through saliva aerosols or nasal discharge from an infected person. Currently, no specific vaccines or treatments exist for coronavirus; however, several attempts are being made to develop possible treatments. Hence, it is important to study the viral structure and life cycle to understand its functionality, activity, and infectious nature. Further, such studies can aid in the development of vaccinations against this virus. Microscopy plays an important role in examining the structure and topology of the virus as well as pathogenesis in infected host cells. This review deals with different microscopy techniques including electron microscopy, atomic force microscopy, fluorescence microscopy as well as computational methods to elucidate various prospects of this life-threatening virus.


Subject(s)
Computational Biology/methods , Coronavirus Infections/virology , Microscopy/methods , SARS Virus/pathogenicity , SARS Virus/ultrastructure , Animals , Chlorocebus aethiops , Host-Pathogen Interactions , Humans , Microscopy/classification , Microscopy, Atomic Force , Microscopy, Electron , Microscopy, Electron, Scanning , Microscopy, Fluorescence , SARS Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
17.
J Gen Virol ; 101(9): 925-940, 2020 09.
Article in English | MEDLINE | ID: covidwho-610420

ABSTRACT

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.


Subject(s)
Betacoronavirus/physiology , SARS Virus/physiology , Virus Replication/physiology , Adaptation, Biological , Animals , Antibodies, Viral/immunology , Betacoronavirus/genetics , Cell Line/ultrastructure , Cell Line/virology , Chlorocebus aethiops , Computational Biology , Conserved Sequence , Cross Reactions , Cytopathogenic Effect, Viral , High-Throughput Nucleotide Sequencing , Humans , Immune Sera/immunology , Kinetics , Mice , Microscopy, Electron , Microscopy, Fluorescence , RNA, Viral/isolation & purification , Rabbits , SARS-CoV-2 , Vero Cells/ultrastructure , Vero Cells/virology
18.
Angew Chem Int Ed Engl ; 59(30): 12417-12422, 2020 07 20.
Article in English | MEDLINE | ID: covidwho-343407

ABSTRACT

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand-receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK-II cells.


Subject(s)
Antiviral Agents/pharmacology , Influenza A virus/drug effects , N-Acetylneuraminic Acid/chemistry , Nanogels/chemistry , Animals , Antiviral Agents/chemistry , Dogs , Influenza A virus/physiology , Inhibitory Concentration 50 , Madin Darby Canine Kidney Cells , Microscopy, Atomic Force , Microscopy, Fluorescence , Virus Internalization/drug effects
19.
J Vet Med Sci ; 82(3): 387-393, 2020 Mar 24.
Article in English | MEDLINE | ID: covidwho-11655

ABSTRACT

Adenosine 5'-triphosphate (ATP), the major energy currency of the cell, is involved in many cellular processes, including the viral life cycle, and can be used as an indicator of early signs of cytopathic effect (CPE). In this study, we demonstrated that CPE can be analyzed using an FRET-based ATP probe named ATP indicator based on Epsilon subunit for Analytical Measurements (ATeam). The results revealed that as early as 3 hr, the virus infected cells showed a significantly different Venus/cyan fluorescent protein (CFP) ratio compared to the mock-infected cells. The ATeam technology is therefore useful to determine the early signs of ATP-based CPE as early as 3 hr without morphology-based CPE by light microscopy, and enables high throughput determination of the presence of microorganisms in neglected samples stored in laboratories.


Subject(s)
Adenosine Triphosphate/analysis , Cytopathogenic Effect, Viral , Fluorescence Resonance Energy Transfer/methods , Viruses/metabolism , Animals , Biosensing Techniques , Cell Line , Green Fluorescent Proteins , Humans , Mammals , Microscopy, Fluorescence , Virus Diseases
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