Name: Procedures voor de identificatie van SARS-CoV-2 ingangsremmers als potentiële antivirale middelen met MLV-gebaseerde pseudovirussen (Video) | JoVE Journal
Uploaded: 2026-02-27T14:10:00.000+00:00
Duration: 11 min 43 s
Description: Leer een geoptimaliseerd protocol voor het genereren van MLV-gebaseerde pseudovirussen met SARS-CoV-2 spike-eiwit voor veilige screening van antivirale ingangsremmers onder BSL-2 omstandigheden.

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</ol>

<table><tbody><tr><td>0.2 &micro;m Low protein-binding syringe filters&nbsp;</td><td>Thermo Scientific</td><td>720-1320</td><td>Syringe Filters (0.2 &mu;m,13 mm, PES, steriele)</td></tr><tr><td>0.25% Trypsin-EDTA 1X solution</td><td>Gibco</td><td>25200-056</td><td>Trypsin-EDTA (0.25%), phenol red</td></tr><tr><td>0.45 &micro;m PVDF Bottle-Top Filter Units&nbsp;</td><td>Avantor</td><td>514-1045</td><td>Bottle-Top Vacuum Filtration Systems (PVDF, 250 ml, 50 mm, 0,45 &micro;m)</td></tr><tr><td>1.5 mL Sterile Microcentrifuge Tubes</td><td>Eppendorf</td><td>30123611</td><td>Safe-Lock&nbsp;Tubes</td></tr><tr><td>100 X Penicillin Streptomycin solution</td><td>Corning</td><td>30-002-CI</td><td>Penicillin-Streptomycin Solution, 100x</td></tr><tr><td>5 X Luciferase assay lysis buffer&nbsp;</td><td>Promega</td><td>E2650</td><td>onderdeel van het Promega Bright-Glo Luciferase Assay System</td></tr><tr><td>50 mL Sterile Conical Tubes</td><td>Corning&nbsp;</td><td>352070</td><td>50 mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile</td></tr><tr><td>6-well Tissue Culture Plates</td><td>Thermo Scientific</td><td>140675</td><td>Cell-Culture Treated Multidishes</td></tr><tr><td>96-well Tissue Culture Plates</td><td>Thermo Scientific</td><td>168055</td><td>Nunc&trade; MicroWell&trade; 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td></tr><tr><td>Arbidol hydrochloride</td><td>Sigma-Aldrich</td><td>SML0860</td><td></td></tr><tr><td>Branched polyethylenimine, 25 kDa</td><td>Sigma-Aldrich</td><td>408727</td><td></td></tr><tr><td>Confocal laser microscope&nbsp;(FV 3000)</td><td>Olympus Co.</td><td>SKU: FV3000</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>DMEM (Dulbecco's Modified Eagle Medium) With: High Glucose, L-glutamine, Phenol Red, Sodium Pyruvate)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140-079</td><td>Heat-inactivated (56&deg;C, 30 min)</td></tr><tr><td>HEK293T-derived cells (Lenti-X 293T Cell Line)</td><td>Takara Bio</td><td>632180</td><td>Highly transfectable subclone van HEK293 cellijn en ondersteunt hoge niveaus van virale eiwitexpressie</td></tr><tr><td>HEPES Buffer</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Highly susceptible cells for infection (293T-ACE2.TMPRSS2 (mCherry) Cell Line)</td><td>BEI Resources</td><td>NR-55293</td><td>HEK293T cellijn gemodificeerd om stabiel ACE2 en TMPRSS2 te produceren</td></tr><tr><td>Hydrochloric acid&nbsp;</td><td>Sigma-Aldrich</td><td>H1758-500ML</td><td></td></tr><tr><td>Inverted phase contrast microscope&nbsp;</td><td>Olympus Co.&nbsp;</td><td>SKU: 2404040005</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516-500ML</td><td></td></tr><tr><td>Luciferase substrate</td><td>Promega</td><td>E2650</td><td>onderdeel van het Bright-Glo Luciferase Assay System</td></tr><tr><td>Microplate reader with luminescence detection</td><td>Agilent BioTek</td><td>BTH1MG</td><td></td></tr><tr><td>MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>Opaque White 96-well Plates</td><td>Corning</td><td>3917</td><td>96-well Solid White Flat Bottom Polystyrene TC-treated Microplates</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM999</td><td></td></tr><tr><td>pcDNA3-EGFP plasmid</td><td>Addgene</td><td>13031</td><td>pcDNA3-EGFP was een gift van Doug Golenbock (Addgene plasmid # 13031 ; http://n2t.net/addgene:13031 ; RRID:Addgene_13031)</td></tr><tr><td>pCMV-MLVgagpol plasmid</td><td>-</td><td>-</td><td>Vriendelijk gedoneerd door Dr. Gary Whittaker&nbsp;</td></tr><tr><td>Phosphate-buffered saline without Ca2+ and Mg2+ (PBS)</td><td>Corning</td><td>21-040-CV</td><td>Phosphate-Buffered Saline, 1X zonder calcium en magnesium</td></tr><tr><td>Promega PureYield Plasmid Maxiprep System</td><td>Promega</td><td>A2393</td><td></td></tr><tr><td>pTG-Luc plasmid</td><td>-</td><td>-</td><td>Vriendelijk gedoneerd door Dr. Gary Whittaker&nbsp;</td></tr><tr><td>pUNO1-SARS-CoV-2 Spike (Wuhan) plasmid</td><td>Invivogen</td><td>p1-spike</td><td></td></tr><tr><td>pVSV-G plasmid</td><td>Addgene</td><td>138479</td><td>pVSV-G was een gift van Akitsu Hotta (Addgene plasmid # 138479 ; http://n2t.net/addgene:138479 ; RRID:Addgene_138479)</td></tr><tr><td>Recombinant Human ACE2-Fc fusion protein</td><td>Invivogen</td><td>fc-hace2</td><td></td></tr><tr><td>Reduced serum medium (Opti-MEM)</td><td>Gibco</td><td>31985-070</td><td>Opti-MEM Reduced Serum Medium</td></tr></tbody></table>

procedures voor de identificatie van sars-cov-2 ingangsremmers als potentiële antivirale middelen met mlv-gebaseerde pseudovirussen

Procedures for the Identification of SARS-CoV-2 Entry Inhibitors as Potential Antivirals using MLV-Based Pseudoviruses

Procedures voor de identificatie van SARS-CoV-2 ingangsremmers als potentiële antivirale middelen met MLV-gebaseerde pseudovirussen

The SARS-CoV-2 virus is an emergent, zoonotic pathogen that has caused a huge burden of disease and mortality around the world since its emergence in ...

procedures-for-identification-sars-cov-2-entry-inhibitors-as

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

A Viral Attachment Assay for Screening of Antiviral Compounds

Drie assays, zoals de cytopathische effect (CPE) gebaseerde bepaling, dosis-respons assay en tijd-van-toevoeging (ToA) assay ontwikkeld, geoptimaliseerd, gevalideerd en toegepast op nieuwe antivirale middelen tegen Bluetongue virus (BTV) identificeren alsmede zoals het bepalen van de mogelijke mechanisme-van-actie (MoA) voor nieuw geïdentificeerde antivirale middelen.

Testsystemen voor de identificatie van nieuwe antivirale middelen tegen Bluetongue virus

Hier presenteren we een protocol dat specifieke stappen van de virale binnenkomst onderzoekt om nieuwe antivirale middelen te identificeren en te evalueren.

Early Viral Entry testsystemen voor de identificatie en evaluatie van antivirale verbindingen

Hier presenteren we een protocol voor het genereren van pseudotyped deeltjes in een BSL-2 omgeving waarin de proteïne van de piek van de hoogpathogene virussen Midden-Oosten respiratoir syndroom en het ernstig acuut respiratoir syndroom coronaviruses. Deze pseudotyped deeltjes bevatten een luciferase verslaggever gen waardoor kwantificering van virus host doelcellen indienststelling.

Productie van Pseudotyped deeltjes te bestuderen van hoogpathogene Coronaviruses in een omgeving van bioveiligheid niveau 2

Het doel van het protocol is het illustreren van de verschillende assays met betrekking tot virale vermelding die kan worden gebruikt om kandidaat-virale invoer remmers te identificeren.

Gebruik van virale vermelding testen en moleculaire docking analyse voor de identificatie van antivirale kandidaten tegen Coxsackievirus A16

Het hier beschreven protocol schetst een snelle en effectieve methode voor het meten van neutraliserende antilichamen tegen het SARS-CoV-2 spike-eiwit door het vermogen van herstellende serummonsters te evalueren om infectie te remmen door een verbeterd groen fluorescerend eiwit-gelabeld vesiculaire stomatitisvirus pseudotyped met spike glycoproteïne.

Detectie van SARS-CoV-2 neutraliserende antilichamen met behulp van high-throughput fluorescerende beeldvorming van pseudovirusinfectie

Dit protocol beschrijft de dynamiek van virale infecties met behulp van luciferase- en fluorescentie-expresserende recombinant (r)SARS-CoV-2 en een in vivo beeldvormingssysteem (IVIS) in K18 hACE2 transgene muizen om de behoefte aan secundaire benaderingen te overwinnen die nodig zijn om SARS-CoV-2-infecties in vivo te bestuderen.

Live beeldvorming en kwantificering van virale infectie bij K18 hACE2 transgene muizen met reporter-expressing recombinant SARS-CoV-2

We bieden een protocol dat algemeen kan worden toegepast op het selecteren van aptameren die alleen binden aan infectieuze virussen en niet aan virussen die niet-infectieus zijn gemaakt door een desinfectiemethode of aan andere soortgelijke virussen. Dit opent de mogelijkheid om de infectiviteitsstatus te bepalen in draagbare en snelle tests.

In vitro selectie van aptameren om infectieuze van niet-infectieuze virussen te onderscheiden

Pseudogetypeerde virussen (PV's) zijn replicatie-defecte virionen die worden gebruikt om gastheer-virusinteracties te bestuderen onder veiligere omstandigheden dan het hanteren van authentieke virussen. Hier wordt een gedetailleerd protocol gepresenteerd dat laat zien hoe SARS-CoV-2 PV's kunnen worden gebruikt om het neutraliserende vermogen van het serum van patiënten na COVID-19-vaccinatie te testen.

Gepseudotypeerde virussen als moleculair hulpmiddel om humorale immuunresponsen tegen SARS-CoV-2 te volgen via neutralisatietest

Dit protocol beschrijft de toepassing van een nieuw hybride alfavirus-SARS-CoV-2-pseudovirus (Ha-CoV-2) als platform voor snelle kwantificering van de besmettelijkheid van SARS-CoV-2-varianten en hun gevoeligheid voor neutraliserende antilichamen.

Toepassing van Ha-CoV-2-pseudovirus voor snelle kwantificering van SARS-CoV-2-varianten en neutraliserende antilichamen

We presenteren een geoptimaliseerd in vitro protocol voor het produceren van SARS-CoV-2-virusachtige deeltjes die het authentieke virus nauwkeurig nabootsen. Deze aanpak maakt het mogelijk om virale infectie-, assemblage- en uitgangsmechanismen te onderzoeken zonder de beperkingen van het vereisen van een laboratorium van bioveiligheidsniveau 3.

Productie van een SARS-CoV-2-virusachtig deeltjessysteem om virale levenscycli in vitro te onderzoeken

Leer een geoptimaliseerd protocol voor het genereren van MLV-gebaseerde pseudovirussen met SARS-CoV-2 spike-eiwit voor veilige screening van antivirale ingangsremmers onder BSL-2 omstandigheden.

Procedures voor de identificatie van SARS-CoV-2 ingangsremmers als potentiële antivirale middelen met MLV-gebaseerde pseudovirussen (Video) | JoVE Journal

Procedures voor de identificatie van SARS-CoV-2 ingangsremmers als potentiële antivirale middelen met MLV-gebaseerde pseudovirussen

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