Identification of human herpesvirus 6 variants A and B by primer-specific real-time PCR may help to revisit their respective role in pathology

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Abstract

Background

Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood.

Objectives

To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens.

Study design

The VS-PCR was adapted from a real-time PCR assay, based on TaqMan® technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27–35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts.

Results

The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders.

Conclusions

We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants.

Introduction

Human herpesvirus 6 (HHV-6) was first isolated from patients with lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS) (Salahuddin et al., 1986). The virus has been thereafter identified as the causative agent of exanthem subitum (Yamanishi et al., 1988). HHV-6 is classified in the Betaherpesvirinae subfamily, along with human cytomegalovirus (HCMV) and HHV-7. HHV-6 isolates are classified into two variants, termed A and B, on the basis of distinct genetic, antigenic and biological characteristics (Aubin et al., 1991).

HHV-6B is detected in saliva and peripheral blood mononuclear cells (PBMCs) from healthy individuals (Gautheret et al., 1995). This variant accounts for the majority of symptomatic primary infections in children (Dewhurst et al., 1993), and it is commonly implicated in infections occurring in patients undergoing stem cell or kidney transplantation (Frenkel et al., 1994, Imbert-Marcille et al., 2000, Yalcin et al., 1994).

Conversely, HHV-6A is rarely identified in patients. Some reports support the hypothesis that HHV-6A exhibits a greater neurotropism than HHV-6B (Borghi et al., 2005, Hall et al., 1998, Portolani et al., 2001). In other respects, HHV-6A has been detected in fetuses with hydrops (Ashshi et al., 2000) and in critically ill immunocompetent individuals (Razonable et al., 2002).

Methods to differentiate HHV-6A from HHV-6B in biological samples were firstly based on restriction fragment length analysis after enzyme digestion, amplification with variant-specific primers, or amplimer hybridization with variant-specific probes (Aubin et al., 1991, Aubin et al., 1994). Nevertheless, those methods were time-consuming, and provided good results mainly when only one variant was implicated. More recently, real-time PCR assays based on the analysis of melting curve features have been developed (Razonable et al., 2002, Safronetz et al., 2003). However, the ability of those methods to detect both variants in the case of mixed infections, especially when one variant is in minority, was not clearly evaluated.

We therefore designed a rapid, sensitive and specific method that would be able to differentiate HHV-6A and HHV-6B, even in the case of a mixed infection with one variant in great minority as compared to the other. This technique was adapted from a previously developed real-time PCR assay, based on TaqMan® technology, used for the quantitation of HHV-6 genome (Gautheret-Dejean et al., 2002). The adaptation consisted in the change of a consensual reverse primer into two variant-specific (VS) primers. This method was then applied to a large pattern of biological specimens from different kinds of patients.

Section snippets

HHV-6 strains

Seven different HHV-6 strains were used for this study: three HHV-6A strains (GS, SIE, TAN) and four HHV-6B strains (HST, MAR, BLE, BLA). All viruses were propagated in phytohemagglutinin-stimulated PBMCs, except for GS and HST which were propagated in HSB-2 and MT4 cell lines, respectively. Viral stocks were constituted and stored at −80 °C, as previously described (Aubin et al., 1994).

Patients and samples

Biological samples from different groups of individuals were retrospectively included in this study:

  • (i)

    Group 1 (

VS-PCR sensitivity

Serial 10-fold dilutions of GS (variant A) and HST (variant B) DNA, ranging from 100,000 copies to one copy per well, were tested to determine the linearity and the sensitivity of both VS-PCR assays. For each point, CT values were measured in triplicate. As exemplified in Fig. 1, a linear regression curve was obtained when plotting CT values against the log10 of the viral DNA input for both VS-PCRs. The sensitivity threshold was estimated as 10 copies/well for the PCR-A, and as 1 copy/well for

Discussion

We have developed a new method, based on TaqMan® technology, for the identification of HHV-6 variants A and B in biological samples. This method was adapted from a previous real-time PCR assay developed for the quantitation of the genome of both HHV-6A and HHV-6B (Gautheret-Dejean et al., 2002). According to the alignment of the nucleotide sequences of the region of interest from seven different HHV-6 strains, the forward primer (Taq1) and the TaqMan® probe (H6S) remained identical, and two new

Acknowledgements

This work was supported by the Association pour la Recherche sur le Cancer (Grant number #4763). We thank Emmanuelle Lenotre from Applied Biosystems for technical assistance for the design of primers.

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