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Methodenartikel

An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

845 weergaven

8 juli 2025

In dit artikel

Samenvatting

Source: Tai, C., et al. Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds. J. Vis. Exp. (2015).

This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows assessment of the impact of the test compound on viral envelope fusion with endosomes.

Protocol

Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. For the purpose of describing the protocols, Gaussia luciferase reporter-tagged HCV is used as a model virus. In the context of the representative results, the compounds chebulagic acid (CHLA) and punicalagin (PUG) are used as candidate antivirals that target viral glycoprotein interactions with cell surface glycosaminoglycans during the early viral entry steps. Heparin, which is known to interfere with the entry of many viruses, is used as a positive control t....

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Resultaten

Table 1: Host cell type for viral infection. The cell type used for each viral infection system described in the representative results is indicated.

VirusCell Type
HCMVHEL
HCVHuh-7.5
DENV-2Vero
MV.......

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Openbaarmakingen

No conflicts of interest declared.

Materialen

Lijst van materialen gebruikt in dit artikel
NaamBedrijfCatalogusnummerOpmerkingen
DMEMGIBCO11995-040
FBSGIBCO26140-079
Penicillin-StreptomycinGIBCO15070-063
Amphotericin BGIBCO15290-018
DMSOSigmaD5879
In vitro toxicology assay kit, XTT-basedSigmaTOX2
PBS pH 7.4GIBCO10010023
Microplate readerBio-Tek Instrument, Inc.ELx800
MicrocentrifugeThermo Scientific75002420
BioLux Gaussia luciferase assay kitNew England BiolabsE3300L
LuminometerPromegaGloMax-20/20
Sodium citrate, dihydrateSigma71402
Potassium chlorideSigmaP5405

Tags

Viral entry-assayvirale fusie-assayhepatitis C-virusluciferase-reporterincubatie bij lage temperatuurfysiologische temperatuurverschuivinginhibitie van endocytosebehandeling met testverbindingbioluminescentiedetectieoogsten van supernatant