A one-step RT-PCR and a flow cytometry method as two specific tools for direct evaluation of human herpesvirus-6 replication

In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The f...

Full description

Saved in:
Bibliographic Details
Published in:Journal of virological methods 2003-03, Vol.108 (2), p.213-222
Main Authors: Andre-Garnier, E, Robillard, N, Costa-Mattioli, M, Besse, B, Billaudel, S, Imbert-Marcille, B.-M
Format: Article
Language:English
Subjects:
Antigens, Viral - analysis
Base Sequence
Biological and medical sciences
Cell Line
Cytometry
DNA Primers - genetics
Flow Cytometry - methods
Fundamental and applied biological sciences. Psychology
Genes, Viral
Herpesvirus 6, Human - genetics
Herpesvirus 6, Human - immunology
Herpesvirus 6, Human - physiology
HHV-6
Humans
Leukocytes, Mononuclear - virology
Microbiology
Replication
Reverse Transcriptase Polymerase Chain Reaction
RNA Splicing
RNA, Messenger - analysis
RNA, Messenger - genetics
RNA, Viral - analysis
RNA, Viral - genetics
RT-PCR
Techniques used in virology
Viral Envelope Proteins - genetics
Virology
Virology - methods
Virus Replication
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The first method consisted of a one-step reverse transcription PCR amplifying a part of the late alternatively spliced U100 gene which encode the gp 82–105 viral glycoprotein. Two extraction methods and two RT-PCR kits were evaluated, leading to the selection of TaKaRa mRNA selective PCR kit. The second procedure consisted in a flow cytometry method to analyze the expression of two late viral HHV-6 antigens using 7C7 and 10G6 monoclonal antibodies. Four fixation permeabilization procedures were compared and the preparation of cells with paraformaldehyde (PFA) 4% was found to be optimal. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This kinetic study confirmed that Mabs recognized late antigens and demonstrate that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms are detectable earlier in the cycle.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(03)00037-5