title: Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.
abstract: BACKGROUND
  Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types.
  
  
  OBJECTIVE
  To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting.
  
  
  METHODS
  Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients.
  
  
  RESULTS
  Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood.
  
  
  CONCLUSIONS
  The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France; Virology Laboratory, Nantes University Hospital, Nantes, France. Electronic address: celine.bressollette@univ-nantes.fr.
authors.forename: Céline
authors.initials: C
authors.lastname: Bressollette-Bodin
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France. Electronic address: vanhanguyen03@yahoo.com.
authors.forename: Thi Van Ha
authors.initials: TV
authors.lastname: Nguyen
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France; Virology Laboratory, Nantes University Hospital, Nantes, France. Electronic address: marina.illiaquer@univ-nantes.fr.
authors.forename: Marina
authors.initials: M
authors.lastname: Illiaquer
authors.affiliation: Virology Laboratory, Nantes University Hospital, Nantes, France. Electronic address: bernard.besse@chu-nantes.fr.
authors.forename: Bernard
authors.initials: B
authors.lastname: Besse
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France. Electronic address: cecile.peltier@univ-nantes.fr.
authors.forename: Cécile
authors.initials: C
authors.lastname: Peltier
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France; Clinical Hematology Department, Nantes University Hospital, Nantes, France. Electronic address: Patrice.chevallier@chu-nantes.fr.
authors.forename: Patrice
authors.initials: P
authors.lastname: Chevallier
authors.affiliation: EA4271, Immunovirology and Genetic Polymorphism, Nantes University, Nantes, France; Virology Laboratory, Nantes University Hospital, Nantes, France. Electronic address: Berthe-marie.imbert@univ-nantes.fr.
authors.forename: Berthe-Marie
authors.initials: BM
authors.lastname: Imbert-Marcille
mesh_terms: D015654:Herpesvirus 6, Human / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012322:RNA Precursors / Q000032:analysis* / Q000235:genetics; D012367:RNA, Viral / Q000097:blood; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019349:Roseolovirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D019562:Viral Load; D014773:Virology / Q000379:methods*
chemical_list: D012322:RNA Precursors; D012367:RNA, Viral
keywords: CV; Chromosomally integrated HHV-6; Cq; HHV-6A/HHV-6B; HSCT; Human herpesvirus 6; PBMCs; Quantitative real time reverse transcriptase PCR; RT-PCR; Stem cell transplantation; Viral transcripts; WB; ciHHV-6; coefficient of variation; hematopoietic stem cell transplantation; human herpesvirus 6 type A/type B; mRNA; messenger Ribonucleic acid; peripheral blood mononuclear cells; quantification cycle; reverse transcriptase polymerase chain reaction; whole blood