We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

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Chronic active Epstein-Barr virus (CAEBV) infection is a systemic Epstein-Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.

The herpes simplex virus type 2 (HSV-2) gene UL14 encodes a 32 kDa protein which is a minor component of the virion tegument and is expressed late in infection. The UL14 protein shows varied localization patterns in HSV-2-infected and singly expressing cells, suggesting the possibility that it is multifunctional. We have investigated the influence of the UL14 protein on the intracellular localization of capsid proteins and DNA cleavage and packaging proteins in coexpressing cells. VP26 is the minor capsid protein ; it binds to hexons of the outer capsid shell and is predominantly cytoplasmic upon sole expression. We have found that VP26 coexpressed with the UL14 protein showed mutual and predominant relocation into the nucleus. At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32 and UL33) that are required for DNA cleavage and packaging. We have found that the UL33 protein, which was also cytoplasmic by sole expression, was relocated to the nucleus upon expression with the UL14 protein, which again seemed to be a result of mutual influence. Coexpression experiments also suggested the possibility of a mutual influence between the UL14 and UL17 proteins, and the UL17 protein and VP26. Our results suggest that the UL14 protein can influence the intracellular localization patterns of a number of proteins belonging to the capsid or the DNA encapsidation machinery.

A simultaneous detection system to quantify HSV, HHV-6, and HHV-7 DNA via multiplex real-time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV-6, and three copies for HHV-7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real-time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV-6, and 1.9% for HHV-7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.