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1.
PLoS Pathog ; 17(2): e1009225, 2021 02.
Article in English | MEDLINE | ID: covidwho-1088773

ABSTRACT

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.


Subject(s)
Drug Discovery , Viral Tropism , Virus Internalization , Virus Replication , /drug therapy , Cathepsins , Cell Line , Drug Development , Endocytosis , Furin , Humans , Serine Endopeptidases
2.
Commun Biol ; 4(1): 197, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1082259

ABSTRACT

In light of the recent accumulated knowledge on SARS-CoV-2 and its mode of human cells invasion, the binding of viral spike glycoprotein to human Angiotensin Converting Enzyme 2 (hACE2) receptor plays a central role in cell entry. We designed a series of peptides mimicking the N-terminal helix of hACE2 protein which contains most of the contacting residues at the binding site, exhibiting a high helical folding propensity in aqueous solution. Our best peptide-mimics are able to block SARS-CoV-2 human pulmonary cell infection with an inhibitory concentration (IC50) in the nanomolar range upon binding to the virus spike protein with high affinity. These first-in-class blocking peptide mimics represent powerful tools that might be used in prophylactic and therapeutic approaches to fight the coronavirus disease 2019 (COVID-19).


Subject(s)
/chemistry , Peptides/pharmacology , /physiology , Amino Acid Sequence , Cell Line , Circular Dichroism , Humans , Peptides/chemical synthesis , Peptides/chemistry , Peptides/metabolism , Protein Binding/drug effects , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/metabolism , Virus Replication/drug effects
3.
Int J Mol Sci ; 22(3)2021 Jan 30.
Article in English | MEDLINE | ID: covidwho-1081098

ABSTRACT

The synthesis of α-fluorinated methyl ketones has always been challenging. New methods based on the homologation chemistry via nucleophilic halocarbenoid transfer, carried out recently in our labs, allowed us to design and synthesize a target-directed dipeptidyl α,α-difluoromethyl ketone (DFMK) 8 as a potential antiviral agent with activity against human coronaviruses. The ability of the newly synthesized compound to inhibit viral replication was evaluated by a viral cytopathic effect (CPE)-based assay performed on MCR5 cells infected with one of the four human coronaviruses associated with respiratory distress, i.e., hCoV-229E, showing antiproliferative activity in the micromolar range (EC50 = 12.9 ± 1.22 µM), with a very low cytotoxicity profile (CC50 = 170 ± 3.79 µM, 307 ± 11.63 µM, and 174 ± 7.6 µM for A549, human embryonic lung fibroblasts (HELFs), and MRC5 cells, respectively). Docking and molecular dynamics simulations studies indicated that 8 efficaciously binds to the intended target hCoV-229E main protease (Mpro). Moreover, due to the high similarity between hCoV-229E Mpro and SARS-CoV-2 Mpro, we also performed the in silico analysis towards the second target, which showed results comparable to those obtained for hCoV-229E Mpro and promising in terms of energy of binding and docking pose.


Subject(s)
Antiviral Agents/chemistry , Coronavirus 229E, Human/metabolism , Dipeptides/chemistry , Ketones/chemistry , A549 Cells , Antiviral Agents/pharmacology , Binding Sites , /virology , Cell Line , /metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , /metabolism , Thermodynamics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Virus Replication/drug effects
4.
PLoS One ; 16(2): e0246803, 2021.
Article in English | MEDLINE | ID: covidwho-1079374

ABSTRACT

Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, administration of the currently available oral formulation results in systemic drug levels that are too low for the inhibition of SARS-CoV-2. We hypothesized that the co-formulation of NIC with an endogenous protein, human lysozyme (hLYS), could enable the direct aerosol delivery of the drug to the respiratory tract as an alternative to oral delivery, thereby effectively treating COVID-19 by targeting the primary site of SARS-CoV-2 acquisition and spread. To test this hypothesis, we engineered and optimized composite particles containing NIC and hLYS suitable for delivery to the upper and lower airways via dry powder inhaler, nebulizer, and nasal spray. The novel formulation demonstrates potent in vitro and in vivo activity against two coronavirus strains, MERS-CoV and SARS-CoV-2, and may offer protection against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to SARS-CoV-2 infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure rapid clinical development and wide-spread utilization.


Subject(s)
Antiviral Agents/administration & dosage , Coronavirus Infections/drug therapy , Muramidase/administration & dosage , Niclosamide/administration & dosage , Administration, Inhalation , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cell Line , Drug Compounding , Drug Delivery Systems/instrumentation , Dry Powder Inhalers , Humans , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/drug effects , Muramidase/pharmacology , Muramidase/therapeutic use , Nasal Sprays , Niclosamide/pharmacology , Niclosamide/therapeutic use , /drug effects
5.
Sci Rep ; 11(1): 3379, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1075252

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a pandemic throughout 2020. Since the virus uses angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry, increment of ACE2 would lead to an increased risk of SARS-CoV-2 infection. At the same time, an association of the ABO blood group system with COVID-19 has also been highlighted: there is increasing evidence to suggest that non-O individuals are at higher risk of severe COVID-19 than O individuals. These findings imply that simultaneous suppression of ACE2 and ABO would be a promising approach for prevention or treatment of COVID-19. Notably, we have previously clarified that histone deacetylase inhibitors (HDACIs) are able to suppress ABO expression in vitro. Against this background, we further evaluated the effect of HDACIs on cultured epithelial cell lines, and found that HDACIs suppress both ACE2 and ABO expression simultaneously. Furthermore, the amount of ACE2 protein was shown to be decreased by one of the clinically-used HDACIs, panobinostat, which has been reported to reduce B-antigens on cell surfaces. On the basis of these findings, we conclude that panobinostat could have the potential to serve as a preventive drug against COVID-19.


Subject(s)
ABO Blood-Group System/metabolism , Histone Deacetylase Inhibitors/pharmacology , Panobinostat/pharmacology , Butyric Acid/pharmacology , /prevention & control , Cell Line , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Humans , Serine Endopeptidases
6.
Cell ; 184(1): 120-132.e14, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064914

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.


Subject(s)
Coronavirus Infections/genetics , Genome-Wide Association Study , /physiology , A549 Cells , Cell Line , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus 229E, Human/physiology , Coronavirus Infections/virology , Coronavirus NL63, Human/physiology , Coronavirus OC43, Human/physiology , Gene Knockout Techniques , HEK293 Cells , Host-Pathogen Interactions/drug effects , Humans , Membrane Proteins/metabolism , Metabolic Networks and Pathways/drug effects , Protein Interaction Mapping
7.
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
8.
Sci Rep ; 11(1): 3, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1065927

ABSTRACT

SARS-CoV-2 induces a muted innate immune response compared to other respiratory viruses. Mitochondrial dynamics might partially mediate this effect of SARS-CoV-2 on innate immunity. Polypeptides encoded by open reading frames of SARS-CoV and SARS-CoV-2 have been shown to localize to mitochondria and disrupt Mitochondrial Antiviral Signaling (MAVS) protein signaling. Therefore, we hypothesized that SARS-CoV-2 would distinctly regulate the mitochondrial transcriptome. We analyzed multiple publicly available RNASeq data derived from primary cells, cell lines, and clinical samples (i.e., BALF and lung). We report that SARS-CoV-2 did not dramatically regulate (1) mtDNA-encoded gene expression or (2) MAVS expression, and (3) SARS-CoV-2 downregulated nuclear-encoded mitochondrial (NEM) genes related to cellular respiration and Complex I.


Subject(s)
/virology , DNA, Mitochondrial/genetics , Mitochondria/genetics , Transcriptome , Cell Line , Humans , Mitochondria/virology
9.
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
10.
Nat Commun ; 12(1): 776, 2021 02 03.
Article in English | MEDLINE | ID: covidwho-1062751

ABSTRACT

The rapid expansion of the COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantage of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates have entered clinical trials with a two-dose immunization regimen. However, the waning antibody response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concerns about a possible short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form that encoded the SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular responses, and conferred a near-complete protection against wild SARS-CoV-2 infection in the lungs of hACE2 transgenic mice. Noticeably, the high levels of neutralizing antibodies in BALB/c mice induced by mRNA-RBD vaccination were maintained for at least 6.5 months and conferred a long-term notable protection for hACE2 transgenic mice against SARS-CoV-2 infection in a sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.


Subject(s)
/immunology , /immunology , /genetics , /immunology , /genetics , Animals , Antibodies, Neutralizing/immunology , /genetics , Cell Line , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology
11.
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
12.
Viruses ; 13(2)2021 02 03.
Article in English | MEDLINE | ID: covidwho-1060766

ABSTRACT

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG's antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus OC43, Human/drug effects , Influenza A Virus, H1N1 Subtype/drug effects , Respiratory Syncytial Virus, Human/drug effects , Thapsigargin/pharmacology , Animals , Antiviral Agents/therapeutic use , Betacoronavirus/physiology , Cell Line , Cell Line, Tumor , Cells, Cultured , Coronavirus OC43, Human/physiology , Endoplasmic Reticulum Stress , Humans , Influenza A Virus, H1N1 Subtype/physiology , Mice , Microbial Sensitivity Tests , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Respiratory Syncytial Virus, Human/physiology , Ribavirin/pharmacology , Thapsigargin/therapeutic use , Virus Replication/drug effects
13.
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
14.
Virol J ; 18(1): 16, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-1059645

ABSTRACT

BACKGROUND: SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. METHODS: To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). RESULTS: The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. CONCLUSIONS: In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/blood , /blood , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus/immunology , Vesicular stomatitis Indiana virus/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Line , Convalescence , Humans , Inhibitory Concentration 50 , Luminescence , Spike Glycoprotein, Coronavirus/genetics
15.
Eur Rev Med Pharmacol Sci ; 25(1): 556-566, 2021 01.
Article in English | MEDLINE | ID: covidwho-1055413

ABSTRACT

A novel coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a current outbreak of infection termed Coronavirus Disease 2019 (COVID-19) by the World Health Organization (WHO). COVID-19 is currently a global pandemic that may cause close to half a billion deaths around the world. Until now, there is no effective treatment for COVID-19. Quinacrine (Qx) has been used since the 1930s as preventive antimalarial compound. It is a recognized small molecule inhibitor of RNA virus replication, with known anti-prion activity, and identified as a potent Ebola virus inhibitor both in vitro and in vivo. Recently, Qx has showed anti-SARS-CoV-2 activity. Herein, we review the potential mechanisms associated with quinacrine as an antiviral compound.


Subject(s)
Antiviral Agents/pharmacology , Quinacrine/pharmacology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , Cell Line , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/prevention & control , Cytokines/immunology , Humans , Mice , Quinacrine/administration & dosage , Quinacrine/adverse effects , Virus Replication/drug effects
16.
Int J Mol Sci ; 22(3)2021 Jan 29.
Article in English | MEDLINE | ID: covidwho-1055071

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global public health emergency. Periodontitis, the most prevalent disease that leads to tooth loss, is caused by infection by periodontopathic bacteria. Periodontitis is also a risk factor for pneumonia and the exacerbation of chronic obstructive pulmonary disease, presumably because of the aspiration of saliva contaminated with periodontopathic bacteria into the lower respiratory tract. Patients with these diseases have increased rates of COVID-19 aggravation and mortality. Because periodontopathic bacteria have been isolated from the bronchoalveolar lavage fluid of patients with COVID-19, periodontitis may be a risk factor for COVID-19 aggravation. However, the molecular links between periodontitis and COVID-19 have not been clarified. In this study, we found that the culture supernatant of the periodontopathic bacterium Fusobacterium nucleatum (CSF) upregulated the SARS-CoV-2 receptor angiotensin-converting enzyme 2 in A549 alveolar epithelial cells. In addition, CSF induced interleukin (IL)-6 and IL-8 production by both A549 and primary alveolar epithelial cells. CSF also strongly induced IL-6 and IL-8 expression by BEAS-2B bronchial epithelial cells and Detroit 562 pharyngeal epithelial cells. These results suggest that when patients with mild COVID-19 frequently aspirate periodontopathic bacteria, SARS-CoV-2 infection is promoted, and inflammation in the lower respiratory tract may become severe in the presence of viral pneumonia.


Subject(s)
/metabolism , Culture Media, Conditioned/chemistry , Cytokines/metabolism , Fusobacterium nucleatum/metabolism , /genetics , /virology , Cell Line , Culture Media, Conditioned/pharmacology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Up-Regulation/drug effects
17.
Int J Mol Sci ; 22(3)2021 Jan 28.
Article in English | MEDLINE | ID: covidwho-1055070

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) was identified as the main host cell receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent infection. In some coronavirus disease 2019 (COVID-19) patients, it has been reported that the nervous tissues and the eyes were also affected. However, evidence supporting that the retina is a target tissue for SARS-CoV-2 infection is still lacking. This present study aimed to investigate whether ACE2 expression plays a role in human retinal neurons during SARS-CoV-2 infection. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids and monolayer cultures derived from dissociated retinal organoids were generated. To validate the potential entry of SARS-CoV-2 infection in the retina, we showed that hiPSC-derived retinal organoids and monolayer cultures endogenously express ACE2 and transmembrane serine protease 2 (TMPRSS2) on the mRNA level. Immunofluorescence staining confirmed the protein expression of ACE2 and TMPRSS2 in retinal organoids and monolayer cultures. Furthermore, using the SARS-CoV-2 pseudovirus spike protein with GFP expression system, we found that retinal organoids and monolayer cultures can potentially be infected by the SARS-CoV-2 pseudovirus. Collectively, our findings highlighted the potential of iPSC-derived retinal organoids as the models for ACE2 receptor-based SARS-CoV-2 infection.


Subject(s)
/genetics , Gene Expression , Induced Pluripotent Stem Cells/cytology , Retina/cytology , /physiology , /metabolism , Cell Culture Techniques , Cell Line , Humans , Induced Pluripotent Stem Cells/metabolism , Organoids/cytology , Organoids/metabolism , Retina/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Virus Internalization
18.
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
19.
Arch Virol ; 166(3): 935-941, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1045234

ABSTRACT

Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.


Subject(s)
/growth & development , Gastroenteritis, Transmissible, of Swine/virology , Porcine epidemic diarrhea virus/growth & development , Transmissible gastroenteritis virus/growth & development , Virus Replication/physiology , Animals , Cell Line , Chlorocebus aethiops , Disease Susceptibility , Fluorescent Antibody Technique, Indirect , Intestine, Small/virology , LLC-PK1 Cells , Swine , Swine Diseases/virology , Vero Cells
20.
Cell Rep ; 34(5): 108699, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1044918

ABSTRACT

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody-Dependent Enhancement , Epitopes/immunology , Adolescent , Adult , Aged , Antibodies, Monoclonal/immunology , Antibodies, Viral/therapeutic use , Antigen-Antibody Reactions , /immunology , Cell Line , Child , Cluster Analysis , Female , Humans , Inhibitory Concentration 50 , Male , Middle Aged , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Young Adult
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