Epidemiology of Epstein–Barr virus, cytomegalovirus, and kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

Abstract

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.

Detection of Viral DNA in Kidney Graft Preservation and Washing Solutions Is Predictive of Posttransplant Infections in Pediatric Recipients

“…Thus, in agreement with their ability to establish latency in B lymphocytes and in monocyte progenitor cells, respectively, EBV and HCMV are probably carried by circulating blood cells when transmitted from the donor to the recipient. Remarkably, the prevalence of EBV and HCMV DNA in preservation and washing solutions was similar to the prevalence of EBV DNA and HCMV DNA in the peripheral blood of healthy blood donors reported in the literature (ie, ∼8%-40% and 1%-3%, respectively [16,17]). …”

Establishment and characterization of a novel Waldenström macroglobulinemia cell line, MWCL-1

“…19 The presence of active viral shedding was determined by growing EBV Ϫ Karpas-422 cells in MWCL-1-conditioned media followed by PCR amplification of EBNA1 in the treated Karpas-422 cells.…”

Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells: Comprehensive Analysis of Gene Expression Profiles

“…The PCR primers for amplification of IE-1/IE-2 genes were used as previously reported. 10 The b-actin gene was used as an internal control. Comparative expression levels of the IE-1/ IE-2 mRNAs were calculated according to expression levels of the b-actin mRNA.…”