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1.
Physiol Rep ; 9(2): e14707, 2021 01.
Article in English | MEDLINE | ID: covidwho-1100461

ABSTRACT

The COVID-19 has originated from Wuhan, China, in December 2019 and has been affecting the public health system, society, and economy in an unheard-of manner. There is no specific treatment or vaccine available for COVID-19. Previous data showed that men are more affected than women by COVID-19, then we hypothesized whether sex hormones could be protecting the female organism against the infection. VERO E6 cells have been commonly used as in vitro model for SARS-CoV-2 infection. In our experimental approach, we have treated VERO E6 cells with 17ß-estradiol to evaluate the modulation of SARS-CoV-2 infection in this cell line. Here we demonstrated that estrogen protein receptors ERα, ERß, and GPER1 are expressed by VERO E6 cells and could be used to study the effects of this steroid hormone. Previous and 24-hours post-infection, cells treated with 17ß-estradiol revealed a reduction in the viral load. Afterward, we found that SARS-CoV-2 infection per se results in ACE2 and TMPRSS2 increased gene expression in VERO E6-cell, which could be generating a cycle of virus infection in host cells. The estrogen treatment reduces the levels of the TMPRSS2, which are involved with SARS-CoV-2 infectiveness capacity, and hence, reducing the pathogenicity/genesis. These data suggest that estrogen could be a potential therapeutic target promoting cell protection against SARS-CoV-2. This opens new possibilities for further studies on 17ß-estradiol in human cell lines infected by SARS-CoV-2 and at least in part, explain why men developed a more severe COVID-19 compared to women.


Subject(s)
Antiviral Agents/pharmacology , Estradiol/pharmacology , /drug effects , /genetics , Animals , /virology , Chlorocebus aethiops , Host-Pathogen Interactions , Receptors, Virus/genetics , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Vero Cells
2.
Methods Mol Biol ; 2273: 131-138, 2021.
Article in English | MEDLINE | ID: covidwho-1092090

ABSTRACT

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.


Subject(s)
/metabolism , Cell Culture Techniques/methods , Epithelial Cells/cytology , Nanoparticles/metabolism , Respiratory Mucosa/cytology , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/virology , Gold , Humans , Metal Nanoparticles/chemistry , Molecular Mimicry/immunology , Nose/virology , Respiratory Mucosa/virology , /pathogenicity , Trachea/virology , Vero Cells , Virus Internalization
3.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Article in English | MEDLINE | ID: covidwho-1087557

ABSTRACT

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mp ro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mp ro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mp ro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.


Subject(s)
Antiviral Agents/pharmacology , Bepridil/pharmacology , Drug Discovery , /drug effects , A549 Cells , Animals , Chlorocebus aethiops , Humans , Molecular Docking Simulation , Molecular Structure , Small Molecule Libraries , Vero Cells
4.
Sci Adv ; 7(7)2021 02.
Article in English | MEDLINE | ID: covidwho-1080728

ABSTRACT

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Mutation, Missense , Spike Glycoprotein, Coronavirus , Amino Acid Substitution , Animals , /genetics , Chile , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Male , /metabolism , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
5.
PLoS Pathog ; 17(2): e1009165, 2021 02.
Article in English | MEDLINE | ID: covidwho-1079380

ABSTRACT

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Convalescence , Spike Glycoprotein, Coronavirus , Adult , Aged , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , /immunology , Chlorocebus aethiops , Cloning, Molecular , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Female , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Male , Middle Aged , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
6.
Sci Rep ; 11(1): 3515, 2021 02 10.
Article in English | MEDLINE | ID: covidwho-1078602

ABSTRACT

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.


Subject(s)
/ultrastructure , Virion/ultrastructure , Animals , Chlorocebus aethiops , Electron Microscope Tomography , Plastics , RNA, Viral , Vero Cells , Virus Replication
7.
Nat Commun ; 12(1): 944, 2021 02 11.
Article in English | MEDLINE | ID: covidwho-1078588

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Antibodies, Viral/administration & dosage , /immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Chlorocebus aethiops , Female , Immunoglobulin G/administration & dosage , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Lung/pathology , Lung/virology , Male , Mice, Inbred C57BL , Mice, Transgenic , /physiology , Seroconversion , Vero Cells , Viral Load
8.
Nat Commun ; 12(1): 961, 2021 02 11.
Article in English | MEDLINE | ID: covidwho-1078585

ABSTRACT

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Subject(s)
/virology , CRISPR-Cas Systems , Genome-Wide Association Study , Host-Pathogen Interactions , Virus Internalization , A549 Cells , /metabolism , Animals , Chlorocebus aethiops , Disease Models, Animal , Endosomes/virology , HeLa Cells , Humans , Mesocricetus , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
9.
Mem Inst Oswaldo Cruz ; 116: e200443, 2021.
Article in English | MEDLINE | ID: covidwho-1076303

ABSTRACT

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.


Subject(s)
Cytopathogenic Effect, Viral , Cytoplasmic Vesicles/virology , Vero Cells/virology , Animals , Chlorocebus aethiops , Endocytosis , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron, Transmission , Nucleocapsid , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Virus Internalization
10.
Nutrients ; 13(2)2021 Feb 09.
Article in English | MEDLINE | ID: covidwho-1069852

ABSTRACT

BACKGROUND: Zinc is an essential micronutrient that impacts host-pathogen interplay at infection. Zinc balances immune responses, and also has a proven direct antiviral action against some viruses. Importantly, zinc deficiency (ZD) is a common condition in elderly and individuals with chronic diseases, two groups with an increased risk for severe severe coronavirus disease 2019 (COVID-19) outcomes. We hypothesize that serum zinc content (SZC) influences COVID-19 disease progression, and thus might represent a useful biomarker. METHODS: We ran an observational cohort study with 249 COVID-19 patients admitted in Hospital del Mar. We have studied COVID-19 severity and progression attending to SZC at admission. In parallel, we have studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) replication in the Vero E6 cell line modifying zinc concentrations. FINDINGS: Our study demonstrates a correlation between serum zinc levels and COVID-19 outcome. Serum zinc levels lower than 50 µg/dL at admission correlated with worse clinical presentation, longer time to reach stability, and higher mortality. Our in vitro results indicate that low zinc levels favor viral expansion in SARS-CoV-2 infected cells. INTERPRETATION: Low SZC is a risk factor that determines COVID-19 outcome. We encourage performing randomized clinical trials to study zinc supplementation as potential prophylaxis and treatment with people at risk of zinc deficiency.


Subject(s)
/blood , Zinc/blood , Aged , Animals , Cell Survival , Chlorocebus aethiops , Cohort Studies , Female , Humans , Male , Middle Aged , Vero Cells , Zinc/administration & dosage , Zinc/pharmacology
11.
Cell ; 184(1): 106-119.e14, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064913

ABSTRACT

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.


Subject(s)
/genetics , Coronavirus Infections/genetics , Coronavirus/physiology , Genome-Wide Association Study , Host-Pathogen Interactions , /physiology , A549 Cells , Animals , Biosynthetic Pathways/drug effects , Cell Line , Chlorocebus aethiops , Cholesterol/biosynthesis , Cholesterol/metabolism , Cluster Analysis , Clustered Regularly Interspaced Short Palindromic Repeats , Common Cold/genetics , Common Cold/virology , Coronavirus/classification , Coronavirus Infections/virology , Gene Knockout Techniques , Host-Pathogen Interactions/drug effects , Humans , Mice , Phosphatidylinositols/biosynthesis , Vero Cells , Virus Internalization/drug effects , Virus Replication
12.
Antiviral Res ; 186: 105012, 2021 02.
Article in English | MEDLINE | ID: covidwho-1064809

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (-) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.


Subject(s)
Antiviral Agents/pharmacology , /physiology , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Bronchi/virology , Cells, Cultured , Chlorocebus aethiops , Eukaryotic Initiation Factor-4A/antagonists & inhibitors , Humans , Respiratory Mucosa/virology , Vero Cells , Viral Load/drug effects , /drug effects
13.
Viruses ; 13(2)2021 02 02.
Article in English | MEDLINE | ID: covidwho-1067781

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.


Subject(s)
Antiviral Agents/pharmacology , Olea , Phenylethyl Alcohol/analogs & derivatives , Plant Extracts/pharmacology , /drug effects , Administration, Topical , Animals , Antiviral Agents/chemistry , Carbohydrates/chemistry , Chlorocebus aethiops , Genome, Viral/drug effects , Glycosylation , Microbial Sensitivity Tests , Phenylethyl Alcohol/administration & dosage , Phenylethyl Alcohol/pharmacology , Phosphoproteins/chemistry , Plant Extracts/chemistry , /physiology , Skin Cream , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Inactivation/drug effects
14.
Sci Adv ; 7(6)2021 02.
Article in English | MEDLINE | ID: covidwho-1066794

ABSTRACT

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.


Subject(s)
/metabolism , Gene Expression , Host Microbial Interactions/genetics , RNA, Messenger/metabolism , Viral Nonstructural Proteins/metabolism , Virulence Factors/metabolism , Active Transport, Cell Nucleus/genetics , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Pore/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transfection , Vero Cells , Viral Nonstructural Proteins/genetics
15.
Sci Adv ; 7(2)2021 01.
Article in English | MEDLINE | ID: covidwho-1066788

ABSTRACT

Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/µl) and exhibited a limit of detection (0.38 copies/µl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.


Subject(s)
/diagnosis , Point-of-Care Testing , Saliva/virology , Smartphone , Animals , CRISPR-Cas Systems , Chlorocebus aethiops , Computer Simulation , Female , Humans , Limit of Detection , Macaca mulatta , Male , Molecular Diagnostic Techniques/instrumentation , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Vero Cells , Viral Load
16.
Elife ; 102021 01 04.
Article in English | MEDLINE | ID: covidwho-1063492

ABSTRACT

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.


Subject(s)
Organoids/virology , Respiratory System/virology , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Amino Acid Motifs , Animals , Cell Fusion , Cell Line, Tumor , Chlorocebus aethiops , Humans , SARS Virus/chemistry , SARS Virus/physiology , Serine Endopeptidases , Vero Cells
17.
Int J Mol Sci ; 22(4)2021 Feb 04.
Article in English | MEDLINE | ID: covidwho-1063411

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Deoxycytidine/analogs & derivatives , /drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , /virology , Cell Line , Chlorocebus aethiops , Deoxycytidine/pharmacology , Drug Therapy, Combination , Humans , Inhibitory Concentration 50 , Vero Cells , Virus Replication/drug effects
18.
Nat Commun ; 12(1): 668, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1062750

ABSTRACT

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.


Subject(s)
/drug therapy , /drug effects , /drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Humans , Indoles/pharmacology , Vero Cells , /metabolism
19.
Viruses ; 13(1)2021 Jan 17.
Article in English | MEDLINE | ID: covidwho-1060230

ABSTRACT

Quinacrine (Qx), a molecule used as an antimalarial, has shown anticancer, antiprion, and antiviral activity. The most relevant antiviral activities of Qx are related to its ability to raise pH in acidic organelles, diminishing viral enzymatic activity for viral cell entry, and its ability to bind to viral DNA and RNA. Moreover, Qx has been used as an immunomodulator in cutaneous lupus erythematosus and various rheumatological diseases, by inhibiting phospholipase A2 modulating the Th1/Th2 response. The aim of this study was to evaluate the potential antiviral effect of Qx against denominated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Vero E6 cells. The cytotoxicity of Qx in Vero E6 cells was determined by the MTT assay. Afterwards, Vero E6 cells were infected with SARS-CoV-2 at different multiplicities of infections (MOIs) of 0.1 and 0.01 in the presence of Qx (0-30 µM) to determinate the half maximal effective concentration (EC50). After 48 h, the effect of Qx against SARS-CoV-2 was assessed by viral cytotoxicity and viral copy numbers, the last were determined by digital real-time RT-PCR (ddRT-PCR). Additionally, electron and confocal microscopy of Vero E6 cells infected and treated with Qx was studied. Our data show that Qx reduces SARS-CoV-2 virus replication and virus cytotoxicity, apparently by inhibition of viral ensemble, as observed by ultrastructural images, suggesting that Qx could be a potential drug for further clinical studies against coronavirus disease 2019 (COVID-19) infection.


Subject(s)
Antiviral Agents/pharmacology , Quinacrine/pharmacology , Virus Replication/drug effects , Animals , Cell Line , Chlorocebus aethiops , Microscopy, Electron, Transmission , Vero Cells , Viral Load/drug effects , Virus Internalization/drug effects
20.
Genes (Basel) ; 12(2)2021 01 29.
Article in English | MEDLINE | ID: covidwho-1055035

ABSTRACT

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.


Subject(s)
/metabolism , /metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , /virology , Cell Line , Chlorocebus aethiops , Computer Simulation , Humans , Pandemics , Protein Binding , Protein Domains , Sequence Deletion , Vero Cells
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