696. Development of Real-Time RT-PCR Assay for Monitoring Active HHV-6 Infection
Session: Abstracts: Virology
Friday, October 22, 2010
Background: Monitoring of active human herpesvirus 6 (HHV-6) infection is important for distinguishing between viral reactivation and latency. Aim of the present study was to develop real-time RT-PCR method for analysis of three classes of HHV-6 gene expressions, which could be useful tool for monitoring active viral infection.

Methods: Three HHV-6 genes (U90; IE gene, U12; E gene, U100; L gene) were selected to design primers and probes for the real-time RT-PCR method. HHV-6 B Z29 infected cord blood cell was used for initial validation analysis, and 10 paired peripheral blood mononuclear cells (PBMCs) samples obtained from patients with primary HHV-6 infection were used to evaluate reliability for clinical use. Primary HHV-6 infection was confirmed by viral isolation and seroconversion of HHV-6 IgG antibody.

Results: A linear relationship was obtained between the targets and cycle threshold values in the three real-time RT-PCR assays. Lower detection limit of all three genes transcripts were 100 copies/reaction. In all three real-time RT-PCR methods, coefficients of variations of the methods were less than 1.5% in inter-assay. The U90 (4.63±3.65copies/103 ß-actin) and U12 (0.89±0.64 copies/103 ß-actin) gene transcripts were detected in all 10 PBMCs samples collected in acute phase of the disease. The U100 gene transcript (0.03±0.05copies/103 ß-actin) was detected in 9 of the 10 acute phase samples. None of the U12 and U100 gene transcripts were detected in PBMCs collected at convalescent phase. The low level of U90 gene transcript was detected in only one convalescent phase sample.

Conclusion: Our results demonstrate the potential clinical application of the real-time RT-PCR assays for monitoring active HHV-6 infection, which could be useful to differentiate between reactivation and latency.

Subject Category: V. Virology including clinical and basic studies of viral infections, including hepatitis

Tetsushi Yoshikawa, MD , Pediatrics, Fujita Health University, Toyoake, Japan
Masaru Ihira, PhD , Faculty of Clinical Engineering, Fujita Health University, Toyoake, Japan
Ken Sugata, MD , Pediatrics, Fujita Health University, Toyoake, Japan
Yoshizo Asano, MD , Pediatrics, Fujita Health University, Toyoake, Japan


T. Yoshikawa, None

M. Ihira, None

K. Sugata, None

Y. Asano, None

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