Generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection
artículo original
Fecha
2021-03-05Autor
Arias Arias, Jorge Luis
Mora Rodríguez, Rodrigo Antonio
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The genus Flavivirus within the family Flaviviridae includes many viral species of medical
importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among
others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of
viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cellbased molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirusactivatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent
protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG).
When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent
protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the
generation, selection and implementation of stable BHK-21 cells expressing our flavivirus geneticallyencoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe
the image analysis procedures and provide the required software pipelines. Our reporter cells allow the
implementation of single-cell infection kinetics as well as plaque assays for both reference and native
strains of flaviviruses by live-cell imaging.
External link to the item
10.21769/BioProtoc.3942Colecciones
- Microbiología [1171]