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 24hpi up to 48hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population.
A flow-FISH assay for the quantitative analysis of parvovirus B19 infected cells / Manaresi, E.; Bua, G.; Bonvicini, F.; Gallinella, G.. - In: JOURNAL OF VIROLOGICAL METHODS. - ISSN 0166-0934. - STAMPA. - 223:(2015), pp. 50-54. [10.1016/j.jviromet.2015.07.013]
A flow-FISH assay for the quantitative analysis of parvovirus B19 infected cells
MANARESI, ELISABETTA;BUA, GLORIA;BONVICINI, FRANCESCA;GALLINELLA, GIORGIO
2015
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 24hpi up to 48hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
