title: A flow-FISH assay for the quantitative analysis of parvovirus B19 infected cells.
abstract: Human parvovirus B19 (B19V) replication is a process highly dependent on the cellular environment, therefore methodologies allowing for analysis at single cell level could represent effective tools to understand cell-to cell differences in the replication process and to investigate cell-virus interactions. Fluorescence in situ hybridization (FISH) can be combined with flow cytometry (flow-FISH) to enable the detection of target nucleic acid sequences in thousands of individual cells in a short amount of time. In the present study, a flow-FISH assay based on the use of a digoxigenin-labeled genomic probe has been developed to discriminate B19V infected cells following in vitro infection of UT7/EpoS1 cell line and EPCs (erythroid progenitor cells) generated from peripheral blood mononuclear cells. In B19V infected UT7/EpoS1 and EPCs, viral nucleic acids were detected by the flow-FISH assay starting from 24 hpi up to 48 hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population.
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, Bologna 40138, Italy. Electronic address: elisabetta.manaresi@unibo.it.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Manaresi
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, Bologna 40138, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Bua
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, Bologna 40138, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bonvicini
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, Bologna 40138, Italy; S. Orsola-Malpighi Hospital - Microbiology, University of Bologna, Via Massarenti 9, Bologna 40138, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gallinella
mesh_terms: D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016732:Parvovirus B19, Human / Q000254:growth & development*; D014773:Virology / Q000379:methods*
keywords: B19V; Flow cytometry; Flow-FISH; Fluorescence in situ hybridization assay; Viral nucleic acids