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1.
J Virol ; 94(15)2020 07 16.
Article in English | MEDLINE | ID: covidwho-690841

ABSTRACT

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Respiratory Mucosa/virology , Severe Acute Respiratory Syndrome/virology , Virus Replication , Animals , Cell Line , Cells, Cultured , Chlorocebus aethiops , Humans , Pandemics , Vero Cells
2.
Int J Mol Sci ; 21(14)2020 Jul 21.
Article in English | MEDLINE | ID: covidwho-670344

ABSTRACT

Coronavirus 2 (CoV) Severe Acute Respiratory Syndrome (SARS-CoV2) is causing a highly infectious pandemic pneumonia. Coronaviruses are positive sense single-stranded RNA viruses that infect several animal species, causing symptoms that range from those similar to the common cold to severe respiratory syndrome. The Angiotensin Converting Enzyme 2 (ACE2) is the SARS-CoV2 functional receptor. Measures are currently undertaken worldwide to control the infection to avoid disruption of the social and economic equilibrium, especially in countries with poor healthcare resources. In a guarded optimistic view, we hope that the undertaken preventive and treatment measures will at least contribute to contain viral diffusion, attenuate activity, or even eliminate SARS-CoV2. In this review, we discuss emerging perspectives for prevention/treatment of COVID-19 infection. In addition to vaccines under development, passive immunization is an open opportunity since patients develop neutralizing antibodies. A full spectrum of potential drugs for COVID-19 infections could in turn affect virus binding or enzymatic activities involved in viral replication and transcription. Furthermore, clinical trials are currently evaluating the safety and efficacy of anti-inflammatory drugs, such as tocilizumab. Bioinformatics may allow characterization of specific CD8+ and CD4+ T cell responses; thus, CoV2 T cells' frequency can be correlated with the disease severity and outcome. Combinatorial antibody phage display may be empowered to identify the immune repertoire of CoV2-specific neutralizing antibodies.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Antiviral Agents/therapeutic use , Coronavirus Infections , Immunization, Passive/methods , Pandemics , Pneumonia, Viral , A549 Cells , Animals , Anti-Inflammatory Agents/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/drug effects , Betacoronavirus/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Chlorocebus aethiops , Coronavirus Infections/diagnosis , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Humans , Lymphocyte Count , Pandemics/prevention & control , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/diagnosis , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
3.
Euro Surveill ; 25(25)2020 06.
Article in English | MEDLINE | ID: covidwho-649992

ABSTRACT

The advent of COVID-19, has posed a risk that human respiratory samples containing human influenza viruses may also contain SARS-CoV-2. This potential risk may lead to SARS-CoV-2 contaminating conventional influenza vaccine production platforms as respiratory samples are used to directly inoculate embryonated hen's eggs and continuous cell lines that are used to isolate and produce influenza vaccines. We investigated the ability of these substrates to propagate SARS-CoV-2 and found that neither could support SARS-CoV-2 replication.


Subject(s)
Chickens/immunology , Coronavirus/physiology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Madin Darby Canine Kidney Cells , Receptors, Virus/metabolism , Virus Cultivation/methods , Virus Replication , Animals , Betacoronavirus , Cell Line , Chickens/virology , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Dogs , Eggs , Humans , Pandemics , Pneumonia, Viral , Severe Acute Respiratory Syndrome
4.
Viruses ; 12(6)2020 05 30.
Article in English | MEDLINE | ID: covidwho-645642

ABSTRACT

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Subject(s)
Antiviral Agents/pharmacology , Drug Discovery/methods , Genes, Reporter/physiology , RNA Viruses/drug effects , Replicon/physiology , Animals , Antiviral Agents/chemistry , Cell Line , Chlorocebus aethiops , Cricetinae , Humans , RNA Viruses/genetics , Transfection , Vero Cells
5.
Genes (Basel) ; 11(7)2020 07 07.
Article in English | MEDLINE | ID: covidwho-640013

ABSTRACT

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , RNA, Long Noncoding/metabolism , Viral Proteins/metabolism , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , Biomarkers/metabolism , Bronchi/cytology , Cell Death , Cell Line , Cluster Analysis , Coronavirus Infections/genetics , Coronavirus Infections/virology , Epithelial Cells/cytology , Epithelial Cells/virology , Gene Regulatory Networks , Humans , Immunity, Innate , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Lung/metabolism , Lung/pathology , Lung/virology , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/virology , RNA, Long Noncoding/genetics , Transcriptome
6.
Int J Mol Sci ; 21(13)2020 Jun 30.
Article in English | MEDLINE | ID: covidwho-635630

ABSTRACT

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.


Subject(s)
Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Protein-Arginine Deiminases/metabolism , Betacoronavirus/isolation & purification , Case-Control Studies , Cell Line , Coronavirus Infections/metabolism , Cytokines/metabolism , Databases, Factual , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Extracellular Vesicles/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Lung/enzymology , Lung/pathology , Lung/virology , Pandemics , Pneumonia, Viral/metabolism , Protein Interaction Maps , Protein-Arginine Deiminases/genetics , RNA, Messenger/metabolism
7.
Fertil Steril ; 114(1): 33-43, 2020 07.
Article in English | MEDLINE | ID: covidwho-634346

ABSTRACT

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Fertility/physiology , Gene Expression Regulation, Viral/physiology , Pneumonia, Viral/metabolism , Reproduction/physiology , Virus Internalization , Adolescent , Adult , Animals , Betacoronavirus/genetics , Cell Line , Coronavirus Infections/genetics , Female , Humans , Macaca fascicularis , Male , Ovary/cytology , Ovary/metabolism , Ovary/virology , Pandemics , Peptidyl-Dipeptidase A/biosynthesis , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/genetics , Pregnancy , Proteomics/methods , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Testis/cytology , Testis/metabolism , Testis/virology , Transcriptome/physiology , Young Adult
8.
Int J Mol Sci ; 21(12)2020 Jun 26.
Article in English | MEDLINE | ID: covidwho-627906

ABSTRACT

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/virology , Evolution, Molecular , Host Microbial Interactions/physiology , Pneumonia, Viral/virology , Receptors, Virus/metabolism , Virus Internalization , Acetylesterase/metabolism , Animals , Betacoronavirus/genetics , Binding Sites , Cell Line , Coronavirus/genetics , Esterases , Gene Transfer, Horizontal , Glycosaminoglycans/metabolism , Hemagglutinins, Viral/genetics , Humans , Lectins/metabolism , Pandemics , Polysaccharides , Receptors, Virus/chemistry , Sialic Acids/chemistry , Sialic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/physiology , Torovirus , Viral Fusion Proteins/genetics
9.
Virol Sin ; 35(3): 311-320, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-617330

ABSTRACT

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury (AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (KPTECs) in 2D and 3D culture systems using conditional reprogramming (CR) and organoids techniques. These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters (SLC34A3 and cubilin). They also expressed angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3D organoids culture compared to that in 2D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike (S) protein was able to enter CR cells with luciferase reporter. This integrated 2D CR and 3D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.


Subject(s)
Betacoronavirus/metabolism , Cell Culture Techniques/methods , Coronavirus Infections/virology , Coronavirus/metabolism , Epithelial Cells/virology , Kidney/virology , Pneumonia, Viral/virology , Animals , Betacoronavirus/pathogenicity , Cell Line , Coronavirus/pathogenicity , DNA Damage , Disease Models, Animal , Humans , Organoids , Pandemics , Peptidyl-Dipeptidase A/metabolism , Receptors, Cell Surface/metabolism , SARS Virus/metabolism , SARS Virus/pathogenicity , Sodium-Phosphate Cotransporter Proteins, Type IIc/metabolism , Spike Glycoprotein, Coronavirus/metabolism
10.
Sci Rep ; 10(1): 10285, 2020 06 24.
Article in English | MEDLINE | ID: covidwho-617064

ABSTRACT

A direct approach to limit airborne viral transmissions is to inactivate them within a short time of their production. Germicidal ultraviolet light, typically at 254 nm, is effective in this context but, used directly, can be a health hazard to skin and eyes. By contrast, far-UVC light (207-222 nm) efficiently kills pathogens potentially without harm to exposed human tissues. We previously demonstrated that 222-nm far-UVC light efficiently kills airborne influenza virus and we extend those studies to explore far-UVC efficacy against airborne human coronaviruses alpha HCoV-229E and beta HCoV-OC43. Low doses of 1.7 and 1.2 mJ/cm2 inactivated 99.9% of aerosolized coronavirus 229E and OC43, respectively. As all human coronaviruses have similar genomic sizes, far-UVC light would be expected to show similar inactivation efficiency against other human coronaviruses including SARS-CoV-2. Based on the beta-HCoV-OC43 results, continuous far-UVC exposure in occupied public locations at the current regulatory exposure limit (~3 mJ/cm2/hour) would result in ~90% viral inactivation in ~8 minutes, 95% in ~11 minutes, 99% in ~16 minutes and 99.9% inactivation in ~25 minutes. Thus while staying within current regulatory dose limits, low-dose-rate far-UVC exposure can potentially safely provide a major reduction in the ambient level of airborne coronaviruses in occupied public locations.


Subject(s)
Antiviral Agents/adverse effects , Betacoronavirus/radiation effects , Disinfection/methods , Ultraviolet Rays/adverse effects , Virus Inactivation/radiation effects , Cell Line , Coronavirus 229E, Human/radiation effects , Coronavirus Infections/radiotherapy , Coronavirus OC43, Human/radiation effects , Humans , Pandemics , Particulate Matter/radiation effects , Pneumonia, Viral/radiotherapy , SARS Virus/radiation effects
11.
Proc Natl Acad Sci U S A ; 117(28): 16587-16595, 2020 07 14.
Article in English | MEDLINE | ID: covidwho-611003

ABSTRACT

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.


Subject(s)
Coronavirus Infections/virology , Disease Models, Animal , Lung/pathology , Pneumonia, Viral/virology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , Cell Line , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/therapy , Cricetinae , Humans , Immunization, Passive , Lung/diagnostic imaging , Lung/virology , Mesocricetus , Pandemics , Pneumonia, Viral/pathology , Ribonucleoproteins/chemistry , Vero Cells , Viral Proteins/chemistry , Virus Replication
12.
Cell Stem Cell ; 27(1): 125-136.e7, 2020 07 02.
Article in English | MEDLINE | ID: covidwho-610467

ABSTRACT

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Organoids/virology , Pneumonia, Viral/virology , Tropism , Animals , Autopsy , Cell Line , Coronavirus Infections/pathology , Hepatocytes/pathology , Hepatocytes/virology , Humans , Induced Pluripotent Stem Cells/virology , Liver/pathology , Mice , Pancreas/pathology , Pancreas/virology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Virus Internalization
13.
Emerg Microbes Infect ; 9(1): 1457-1466, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-599993

ABSTRACT

Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Genome, Viral , Pneumonia, Viral/virology , Animals , Betacoronavirus/classification , Betacoronavirus/isolation & purification , Cell Line , Chlorocebus aethiops , Haemophilus parainfluenzae/isolation & purification , Humans , Middle East , Open Reading Frames , Pandemics , Phylogeny , RNA, Viral , Sequence Deletion , Taiwan , Travel , Vero Cells , Virus Cultivation , Whole Genome Sequencing
14.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-595042

ABSTRACT

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Endoribonucleases/metabolism , Exoribonucleases/metabolism , Interferons/antagonists & inhibitors , Interferons/metabolism , Methyltransferases/metabolism , Pneumonia, Viral/metabolism , RNA Helicases/metabolism , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolism , Betacoronavirus/genetics , Cell Line , Coronavirus Infections/genetics , Coronavirus Infections/virology , Endoribonucleases/genetics , Exoribonucleases/genetics , Host-Pathogen Interactions , Humans , Interferons/genetics , Methyltransferases/genetics , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/virology , RNA Helicases/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
15.
Viruses ; 12(6)2020 06 10.
Article in English | MEDLINE | ID: covidwho-592497

ABSTRACT

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.


Subject(s)
Anticoagulants/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Guanidines/pharmacology , Pneumonia, Viral/drug therapy , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Virus Internalization/drug effects , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Betacoronavirus/metabolism , Cell Line , Chlorocebus aethiops , Coronavirus Infections/virology , Gabexate/analogs & derivatives , Gabexate/pharmacology , HEK293 Cells , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
16.
Biochem Biophys Res Commun ; 529(2): 257-262, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-591648

ABSTRACT

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.


Subject(s)
Bombyx/cytology , Bombyx/virology , Nucleopolyhedroviruses/genetics , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/isolation & purification , Animals , Bombyx/enzymology , Cell Line , Cloning, Molecular , Furin/metabolism , Nucleopolyhedroviruses/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
17.
PLoS Biol ; 18(6): e3000715, 2020 06.
Article in English | MEDLINE | ID: covidwho-574821

ABSTRACT

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.


Subject(s)
Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus/classification , Coronavirus/physiology , Endoplasmic Reticulum/pathology , Endoplasmic Reticulum/virology , Virus Replication , Animals , Betacoronavirus/genetics , Betacoronavirus/physiology , Cell Line , Chlorocebus aethiops , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Humans , Middle East Respiratory Syndrome Coronavirus/physiology , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , RNA, Viral/metabolism , Vero Cells
18.
Antimicrob Agents Chemother ; 64(8)2020 07 22.
Article in English | MEDLINE | ID: covidwho-574704

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic that originated in Wuhan, China, in December 2019 has impacted public health, society, the global economy, and the daily lives of billions of people in an unprecedented manner. There are currently no specific registered antiviral drugs to treat or prevent SARS-CoV-2 infections. Therefore, drug repurposing would be the fastest route to provide at least a temporary solution while better, more specific drugs are being developed. Here, we demonstrate that the antiparasitic drug suramin inhibits SARS-CoV-2 replication, protecting Vero E6 cells with a 50% effective concentration (EC50) of ∼20 µM, which is well below the maximum attainable level in human serum. Suramin also decreased the viral load by 2 to 3 logs when Vero E6 cells or cells of a human lung epithelial cell line (Calu-3 2B4 [referred to here as "Calu-3"]) were treated. Time-of-addition and plaque reduction assays performed on Vero E6 cells showed that suramin acts on early steps of the replication cycle, possibly preventing binding or entry of the virus. In a primary human airway epithelial cell culture model, suramin also inhibited the progression of infection. The results of our preclinical study warrant further investigation and suggest that it is worth evaluating whether suramin provides any benefit for COVID-19 patients, which obviously requires safety studies and well-designed, properly controlled randomized clinical trials.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Suramin/pharmacology , Virus Replication/drug effects , Animals , Cell Line , Chlorocebus aethiops , Drug Evaluation, Preclinical , Drug Repositioning , Humans , Pandemics , Vero Cells , Viral Load/drug effects
19.
Trends Pharmacol Sci ; 41(8): 513-517, 2020 08.
Article in English | MEDLINE | ID: covidwho-457060

ABSTRACT

Basic research on SARS-CoV-2 is essential to understand its detailed pathophysiology and identify best drug targets. Models that can faithfully reproduce the viral life cycle and reproduce the pathology of COVID-19 are required. Here, we briefly review the cell lines, organoids, and animal models that are currently being used in COVID-19 research.


Subject(s)
Betacoronavirus/isolation & purification , Biomedical Research/methods , Coronavirus Infections/physiopathology , Pneumonia, Viral/physiopathology , Animals , Cell Line , Coronavirus Infections/virology , Disease Models, Animal , Humans , In Vitro Techniques , Organoids/virology , Pandemics , Pneumonia, Viral/virology
20.
Int J Infect Dis ; 97: 7-10, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-437018

ABSTRACT

OBJECTIVES: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. METHODS: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. RESULTS: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 µM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 µM). CONCLUSIONS: Overall, the data do not support the use of DRV for the treatment of COVID-19.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections , Darunavir/therapeutic use , Pandemics , Pneumonia, Viral , Antiviral Agents/therapeutic use , Cell Line , Cell Survival/drug effects , Coronavirus Infections/drug therapy , Humans , Pneumonia, Viral/drug therapy
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