Development of Reverse Transcriptase PCR Assays for Detection of Active Human Herpesvirus 6 Infection

Abstract

We developed reverse transcriptase (RT) PCR assays for the detection of mRNA from three spliced genes of human herpesvirus 6 (HHV-6), the immediate-early genes U16/U17 and U89/U90 and the late gene U60/U66. Sequence analysis determined the splicing sites of these genes. The new assays may be instrumental in investigating the association between HHV-6 and disease.Human herpesvirus 6 (HHV-6) is the causative agent of exanthema subitum (10). High HHV-6 viral loads have been demonstrated in the settings of allograft rejection, immunodeficiencies, malignancies, and multiple sclerosis, although an etiological correlation is still uncertain (1). This is primarily due to the lack of sensitive diagnostic tools specific for active infection. PCR detection of viral DNA in plasma has been proposed as a marker of active infection, but its sensitivity is low (9). The aim of the present work was to develop HHV-6 reverse transcriptase (RT)-PCR assays, since the expression of viral mRNA constitutes an unambiguous marker of active infection. To discriminate mRNA from genomic DNA, we focused on the detection of spliced gene expression; here we report on RT-PCR assays for the immediate-early genes U16/ U17 and U89/U90 and the late gene U60/U66, as well as on the splicing patterns of these genes for both HHV-6A and HHV-6B.HHV-6A(GS) strain was propagated in the human T-cell line HSB-2; HHV-6B(PL1) strain was propagated in activated peripheral blood mononuclear cells (PBMC). mRNA was extracted using oligo(dT) 25 -coated magnetic beads (Dynabeads mRNA direct kit; Dynal, Oslo, Norway), and total RNA was extracted using the Qiagen RNeasy minikit (Qiagen, Hilden, Germany). Primers (Table 1) were designed based on the HHV-6(U1102) sequence (4).Poly(A) ϩ RNA was treated with the Access RT-PCR kit (Promega, Madison, Wis.). Except for primers C1bis and C2bis, initial denaturation (94°C for 2 min) was followed by 35 amplification cycles (94°C for 1 min, 50°C for 1 min, and 72°C for 2 min) and by final elongation (72°C for 10 min). For primers C1bis and C2bis, initial denaturation (95°C for 15 min) was followed by 40 amplification cycles (95°C for 20 s, 50°C for 45 s, and 72°C for 30 s) and by final elongation (72°C for 2 min).Total RNA samples were treated (35°C for 15 min) with RNase-free DNase (Ambion, Austin, Tex.) and then subjected to reverse transcription (36°C for 90 min) using Moloney murine leukemia virus RT (Promega). One-tenth of the RT reaction mixture was added to 50 pmol of each primer, 100 M each deoxynucleoside triphosphate, 2.5 mM MgCl 2 , 1ϫ Perkin-Elmer buffer II, and 1.25 U of Taq polymerase (Applied Biosystems, Foster City, Calif). Initial denaturation (95°C for 15 min) was followed by 40 amplification cycles (95°C for 20 s, 60°C [50°C for primers C1bis and C2bis] for 45 s and 72°C for 30 s) and by final elongation (72°C for 2 min).For sequencing, amplimers were purified using the Qiaquick spin extraction kit (Qiagen), amplified, and sequenced with the primers mentioned above (Table 1; Fig. 1). The nucleotide positions of...

Selective suppression of IL-12 production by human herpesvirus 6

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Detection and Typing of Human Herpesvirus 6 by Molecular Methods in Specimens from Patients Diagnosed with Encephalitis or Meningitis

“…Since HHV-6 has the ability to persist and establish latency, the problem arises that diagnostic tests must be able to distinguish active viral replication from latent infection. Reverse transcription-PCR assays have been developed to detect the presence of viral mRNA, which constitutes a marker for active infection (44). The detection of immunoglobulin M or immunoglobulin G antibodies to HHV-6 in serum or CSF does not discriminate between active infection and latent/chronic persistent infection.…”

Update on Human Herpesvirus 6 Biology, Clinical Features, and Therapy

“…Virus isolation and cocul- tivation with PBMCs or CBMCs, although highly indicative of active infection, is less sensitive and is a difficult and timeconsuming procedure. PCR on small amounts of PBMCs has been suggested to discern active from latent infection (150), as well as serum-plasma PCR (170,317) and reverse transcription-PCR (311,416,444), which allow detection of circulating viral genomes and viral mRNAs, respectively. More recently, however, quantitative PCR analysis, which allows fast, sensitive, and absolute quantitation of viral load, has become the method of choice (20,135,176,394).…”