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mSphere ; 6(6): e0071121, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1546463


The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N6-(Δ2-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 µM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.

Antiviral Agents/pharmacology , Bacteria/metabolism , Culture Media/chemistry , Metabolic Networks and Pathways , Microbiota/physiology , SARS-CoV-2/drug effects , Symbiosis/physiology , Bacteria/chemistry , Bacteria/classification , Bacteria/growth & development , Biological Assay , Cell Line, Tumor , Culture Media/pharmacology , Humans , Molecular Docking Simulation , Protease Inhibitors/pharmacology , Protein Binding
Viruses ; 12(11)2020 10 23.
Article in English | MEDLINE | ID: covidwho-895403


Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore® Molecular Transport Medium (MTM) and COPAN eNAT™ completely inactivate high-titre SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent.

Betacoronavirus/isolation & purification , Point-of-Care Testing , Specimen Handling , Virus Inactivation , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Culture Media/analysis , Culture Media/pharmacology , Humans , Point-of-Care Testing/standards , SARS-CoV-2 , Specimen Handling/methods , Specimen Handling/standards , Viral Load/drug effects , Virus Inactivation/drug effects
Cytotherapy ; 22(8): 458-472, 2020 08.
Article in English | MEDLINE | ID: covidwho-209852


BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.

Blood Platelets/cytology , Cell Culture Techniques/methods , Filtration , Mesenchymal Stem Cells/cytology , Nanotechnology , Adipogenesis/drug effects , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Culture Media/pharmacology , Extracellular Vesicles/metabolism , Gene Expression Profiling , Humans , Immunophenotyping , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Particle Size , Serum/chemistry