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 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.
Andre-Garnier
E
E
Virology Laboratory EA-1156, Institute of Biology, Nantes University Hospital, 9 quai Moncousu, F-44093 Nantes cedex, France.
Robillard
N
N
Costa-Mattioli
M
M
Besse
B
B
Billaudel
S
S
Imbert-Marcille
B-M
BM
D000956:Antigens, Viral / Q000032:analysis; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005814:Genes, Viral; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D000956:Antigens, Viral; D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral; D014759:Viral Envelope Proteins