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.

Objectives: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting.

Study-design: 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.

Conclusion: 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.

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.