In addition, we include the optimized CellProfiler analysis pipelines for studies employing this or other cell-based reporters. Furthermore, we provide a rationale for a software-based image analysis approach to demonstrate the capabilities of this reporter cell line for single-cell and viral-plaque tracking. Based on our recently published flavivirus-activatable GFP (FlaviA-GFP) and flavivirus-activatable mNeptune (FlaviA-mNeptune) reporters (Arias-Arias et al., 2020), here we describe in detail a protocol for the generation, selection, and implementation of stably-transduced reporter BHK-21 cells for live imaging of flavivirus infection in single cells and viral plaques. In this context, cell-based molecular reporters of the flaviviral proteases constitute a favorable alternative for the study of native flavivirus strains by live-cell imaging. Reporter replicons and viral genomes are suitable for live-cell imaging assays, but they are restricted to particular molecular clones mainly derived from reference strains and thus, not applicable when working with clinical isolates or native viral strains. Immunolabeling implies both fixation and permeabilization which hamper their implementation for studies in living cells. At present, the detection of flavivirus-infected cells is based on either the antibody labeling of viral proteins (Balsitis et al., 2008), the use of recombinant reporter replicons and viral genomes (Li et al., 2013 Schmid et al., 2015 Xie et al., 2016 Tamura et al., 2017 Kümmerer, 2018), or the application of genetically-encoded molecular reporters of the flavivirus NS2B-NS3 proteolytic activity (Medin et al., 2015 Hsieh et al., 2017 McFadden et al., 2018). Keywords: Flavivirus ( 黄病毒属), Fluorescence ( 荧光), NS2B-NS3 ( NS2B-NS3), Protease ( 蛋白酶), Live-cell imaging ( 活细胞成像), Reporter cells ( 报告细胞), Plaque assay ( 噬斑试验), Image analysis ( 图象分析)įlaviviruses represent an emerging and re-emerging global threat that cause diseases both in animals and humans, including many medically relevant viruses like yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DENV), and Zika virus (ZIKV), among others (Gould and Solomon, 2008). Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection. 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. We also describe the image analysis procedures and provide the required software pipelines. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Among the latter, our flavivirus-activatable 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). 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 cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. 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. A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.J Biol Chem 295(8): 2212-2226. Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection. Readers should cite both the Bio-protocol article and the original research article where this protocol was used:Arias-Arias, J. Updated Version Updated Version 引用 收藏 提问与回复 分享您的反馈 Cited by
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