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.
Subject(s)
Cytomegalovirus/isolation & purification , Herpesviridae Infections/epidemiology , Herpesvirus 4, Human/isolation & purification , Herpesvirus 8, Human/isolation & purification , Leukocytes/virology , Polymerase Chain Reaction/methods , Blood Donors , Cell Line , Cytomegalovirus/genetics , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/epidemiology , Cytomegalovirus Infections/virology , DNA, Viral/analysis , Epstein-Barr Virus Infections/diagnosis , Epstein-Barr Virus Infections/epidemiology , Epstein-Barr Virus Infections/virology , Herpesviridae Infections/diagnosis , Herpesviridae Infections/virology , Herpesvirus 4, Human/genetics , Herpesvirus 8, Human/genetics , Prevalence , Sarcoma, Kaposi/diagnosis , Sarcoma, Kaposi/epidemiology , Sarcoma, Kaposi/virology , Sensitivity and SpecificityABSTRACT
We evaluated the cytotoxic and apoptotic effects of two purine nucleoside analogues, acyclovir (ACV) and ganciclovir (GCV), on lymphoma cells stably harboring Kaposi's sarcoma-associated herpesvirus (KSHV). Colorimetric caspase assay, flow cytometry, and immunoblotting with antibodies against apoptosis-related molecules revealed that GCV has cytotoxic activity toward KSHV-infected primary effusion lymphoma cells, while ACV has weak or little activity. In addition to the GCV-induced cytotoxicity, apoptosis via caspase-7/8, cleavage of poly(ADP-ribose) polymerase, and accumulation of p53 and p21 were induced by GCV treatment. In contrast, neither ACV nor GCV have cytotoxicity- or apoptosis-inducing activities toward uninfected cells.
