Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo

Abstract

Out of 64 cases of non-Hodgkin's lymphomas (NHL), 55 cases of Hodgkin's disease (HD) and 31 cases of multiple sclerosis (MS), 2 NHL, 7 HD and 1 MS cases were found positive by polymerase chain reaction (PCR) for the presence of HHV-6 sequences in pathologic lymph nodes of the lymphomas and in peripheral blood mononuclear cells (PBMCs) of MS. A further analysis of the PBMCs of the PCR positive cases by standard Southern blot technique revealed only 2 NHL, 3 HD and 1 MS cases as positive, indicating that these six patients have an unusually high viral copy number in the PBMCs. Restriction analysis, carried out using probes representative of different regions of the virus, showed that three cases retain only a deleted portion of the viral genome. In the remaining three cases a complete viral genome was present, containing the right end sequences in which the rep-like gene, possibly crucial to the viral and cellular life cycle, is located. The analysis by pulsed field gel electrophoresis (PFGE) of the total DNA of the PBMCs obtained directly, without culture from PBMCs of these last three cases (1 NHL, 1 HD, and 1 MS), using the same probes, showed the absence of free viral molecules and the association of viral sequences with high molecular weight DNA. These results are consistent with in vivo integration of the entire virus in the cellular genome. A further study of the same patients with chromosome fluorescence in situ hybridization (FISH) showed in all the three cases the presence of a specific hybridization site, located at the telomeric extremity of the short arm of chromosome 17 (17p13), suggesting that this location is at least a preferred site of an infrequent, but possibly biologically important, integration phenomenon.

Expression of Human Herpesvirus-6 Antigens in Benign and Malignant Lymphoproliferative Diseases

“…In one NHL case and in two HD cases, the viral copy number was so high that it was detectable also by Southern blot analysis. 22,30 Genotype characterization, performed by a PCR assay, 26 showed the HHV-6 variant B genome in all cases, with the exception of three AILD cases, showing HHV-6 variant A genome in two cases and a mixture of HHV-6 variant A and B genomes in one case, as previously reported. 26 Furthermore, tissues from five NHL, four HD, and five reactive lymphadenopathy cases, which tested negative for HHV-6 sequences by PCR, were also included in the study to serve as negative controls in immunohistochemical experiments.…”

“…One of these cases was also positive by Southern blot analysis, indicating high copy number latent integration of the HHV-6 genome, as previously reported. 30 Neoplastic cells were consistently negative for HHV-6 antigens in all NHL tissues examined, even in the B-NHL case (follicular center type) with so a high copy number that it tested positive for HHV-6 DNA by Southern blot analysis. Cytoplasmic staining was observed with the antibody p101K in rare plasma cells interspersed among neoplastic cells and in some isolated spindle-shaped stromal cells located in the lymph node capsule and in the surrounding fibroadipose tissue.…”

“…22 One NHL, 30 two HD, 22 and the five AILD 26 cases have been previously described. In one NHL case and in two HD cases, the viral copy number was so high that it was detectable also by Southern blot analysis.…”

Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis

“…However, when the cell count was at its lowest, i.e., Ͻ1 ϫ 10 3 cells/ml, only half of the samples tested contained detectable HHV-6 DNA, presumably because our test was at the limit of sensitivity. HHV-6 DNA in CSF is usually taken to indicate active infection, but in chromosomal viral integration, there is no evidence to date of virus replication (27,38). Thus, many of our patients with integrated virus were initially given a misdiagnosis of HHV-6 encephalitis because of viral DNA in CSF, although they had disparate symptoms, final diagnoses, and outcomes, including 2 patients with confirmed cases of herpes simplex encephalitis, one of whom died (Table 2).…”

“…However, this interpretation has been questioned in view of the phenomenon of HHV-6 chromosomal integration (8,46). HHV-6 is the only human herpesvirus found integrated into host chromosomes (10,37,38). The occasional individual with such integration is easily identifiable, since every leukocyte inevitably contains viral sequences and there are thus characteristically persistent high levels of HHV-6 DNA in both serum and whole blood (8,46).…”