In vitro activation of human herpesviruses 6 and 7 from latency

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.     

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6B's in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.     

Chronic myelopathy associated with human herpesvirus-6

Human herpesvirus-6 (HHV-6) is implicated in a variety of neurologic diseases. We report a previously healthy elderly woman with progressive spastic paraparesis. At autopsy the spinal cord showed widespread demyelination, axonal loss, and chronic inflammation. HHV-6 DNA was amplified, using polymerase chain reaction, from spinal cord tissue, and glial cells were immunoreactive with an HHV-6 antibody. These findings suggest that HHV-6 may cause myelopathy.     

Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line

Human herpesvirus 6 (HHV-6) genome has been found in several human lymphoid malignancies, but configuration of the HHV-6 genome has not been well delineated. We established the HHV-6-positive, Epstein-Barr virus-negative Burkitt's lymphoma cell line Katata. In this study we investigated the status of the HHV-6 genome in Katata cells. Neither linear nor circular HHV-6 DNA was detected by Gardella gel analysis. The fluorescence in situ hybridization technique enabled us to directly visualize the integrated HHV-6 DNA at the single-cell level. Only one integrated site of viral DNA was detected in metaphase chromosomes and it was preferentially located at the long arm of chromosome 22 (22q13). Treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with calcium ionophore A23187 led to induction of the HHV-6 immediate-early gene as well as the late gene. Sodium n-butyrate also gave rise to expression of the HHV-6 genes. The TPA inducibility was synergistically enhanced when combined with A23187 or n-butyrate. Our study provides, for the first time, an in vitro model system of latent HHV-6 infection whose genome is integrated into host DNA of lymphoma cells.     

In vitro generation of human dendritic cells and cell therapy

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.     

Optociliary veins in optic nerve sheath meningioma. Indocyanine green videoangiography findings

Human herpesvirus 6 (HHV-6) DNA levels in peripheral blood mononuclear cells were prospectively evaluated in 20 cytomegalovirus-seronegative allogeneic marrow transplant patients and in 10 healthy control subjects. Blood and saliva specimens obtained weekly for 3 months after transplant were evaluated by quantitative HHV-6 polymerase chain reaction. One of 20 patients experienced primary HHV-6 infection after marrow transplant (seroconversion, HHV-6 viremia, skin rash); 18 of 20 had increased peripheral blood mononuclear cell HHV-6 DNA levels consistent with asymptomatic reactivations, and 1 patient experienced a reactivation-associated skin rash. Genotyping revealed HHV-6 variant B DNA in all cases. Therapy with acyclovir or intravenous immunoglobulin was not correlated with lower HHV-6 DNA levels. Thus, asymptomatic HHV-6 reactivations appear to be common following allogeneic marrow transplantation. Among HHV-6-seronegative and viral DNA-negative patients, primary HHV-6 infection can ensue in association with self-limited clinical symptoms, including diffuse maculopapular rash.     

Intervening to reduce anxiety for women with mild dyskaryosis: do we know what works and why?

Telomeric repeat sequences (TRS) have been identified close to, but not at, the genome termini of several lymphotropic herpesviruses, including human herpesviruses 6 and 7 (HHV-6, HHV-7). The functional significance of these motifs remains uncertain. Since telomeric sequences can mediate stable retention of episomal DNA in yeast, we have tested whether the TRS motifs from HHV-6 might mediate a similar function in human cells. Several candidate sequences were assessed for their ability to provide nuclear retention to an autonomously replicating vector in rapidly dividing human tissue culture cells, including HHV-6 TRS DNA, as well as telomeric DNA from human cells and sequences from Epstein-Barr virus (EBV). However, only a vector carrying the EBV-derived retention mechanism showed a significant level of nuclear retention. Neither the HHV-6 TRS motifs, nor human telomeric sequences, mediated nuclear retention of episomal DNA in human cells.     

Delta-opioid ligands reverse alfentanil-induced respiratory depression but not antinociception

Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin's lymphomas, 14 Hodgkin's disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called "mummified" Reed-Sternberg cells, in two Hodgkin's disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder.     

Human herpesvirus-6 meningoencephalitis in a recipient of an unrelated allogeneic bone marrow transplantation

BACKGROUND: Human herpesvirus-6 (HHV-6) has been implicated in bone marrow suppression, interstitial pneumonitis, and fatal meningoencephalitis in bone marrow transplant (BMT) recipients. METHODS: We describe the case of a woman with acute myeloid leukemia in second remission who developed febrile meningoencephalitis 8 months after a second unrelated BMT. RESULTS: Computed tomography and magnetic resonance images of the brain were nonspecific. Analysis of cerebrospinal fluid (CSF) revealed lymphocytosis and an increased protein level. Using polymerase chain reaction methods, HHV-6 was the only pathogen detected in CSF, peripheral blood mononuclear cells, and bone marrow. The patient was treated with ganciclovir and foscarnet for 3 months. All clinical manifestations resolved and HHV-6 polymerase chain reaction analysis of CSF became negative 40 days after the beginning of antiviral treatment. CONCLUSIONS: This observation strongly suggests that HHV-6 should be sought in BMT patients with neurological complications and that HHV-6 meningoencephalitis may respond to ganciclovir and foscarnet therapy.     

Thoracic esophageal diverticula. Why is operation necessary?

Human herpesvirus 6 (HHV-6) is a human herpesvirus isolated from patients with various lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS). The prevalence of HHV-6 infection and its correlation as a cofactor in pathogenicity of HIV infection was investigated in serum samples from 365 healthy volunteers at various age groups, 50 persons at risk for HIV-1 infection, and 90 HIV-1 seropositive individuals. Sera were screened and titrated for antibodies against HHV-6 by a standard indirect immunofluorescence assay on an acetone fixed HHV-6 infected HSB2 cells. The data show high prevalence of HHV-6 in Thailand (71.7%) and the infection is acquired early in life. Prevalence of anti-HHV-6 IgG antibodies was not strikingly different among people at risk for HIV infection, asymptomatic HIV-1 infected cases, and aged-matched controls with low risk for HIV-1 infection. The AIDS cases showed high titers of anti-HHV-6 IgG antibody and high rates for presence of anti-HHV-6 IgM antibody (33.3%) which suggests higher prevalence of HHV-6 infection by either reactivation of an earlier HHV-6 infection or a new primary infection.     

Presence of human herpesvirus 6 variants A and B in saliva and peripheral blood mononuclear cells of healthy adults

Saliva and peripheral blood mononuclear cells (PBMCs) from 44 healthy young adults were tested for human herpesvirus 6 variants A and B (HHV-6A and -6B) DNA by a sensitive nested PCR. HHV-6B infection was ascertained in 98% of the subjects, and 95% were found to excrete variant B in their saliva. HHV-6A was found in the PBMCs of 16%, but was not detected in saliva samples.     

Human Herpesvirus-6 (HHV-6) infection in multiple sclerosis: a preliminary report

We examined cerebral spinal fluid (CSF) from multiple sclerosis (MS) patients and patients with other neurological diseases (OND) for antibody specific for Human Herpesvirus-6 (HHV-6) and for HHV-6 DNA detectable by PCR. CSF from MS patients had a higher frequency of IgG antibody to HHV-6 late antigens (39.4%) compared with CSF from OND (7.4%). In contrast, the frequency of detectable IgG antibody in CSF from MS patients specific for Epstein-Barr Virus (EBV) (12.1%) and Human Cytomegalovirus (HCMV) (6.1%) was much lower. Two of 12 MS CSFs (16.7%) also contained HHV-6 DNA detected by PCR. None of four OND CSF were positive for HHV-6 DNA. Plasma from 16 patients with MS, eight with OND and 72 healthy donors were tested for antibodies by ELISA to HHV-6 early (p41/38) and late (gp110) proteins. Although no differences in anti-gp110 IgG antibody were detected between MS patients, patients with other neurological diseases, and normals, IgG antibody to early protein p41/38 was detected in > 68% of the plasma from MS patients, 12.5% from OND patients and 27.8% of the controls. IgM antibody to p41/38 was present in > 56% of MS patients, 12.5% of OND patients, and 19% of controls. These data suggest that more than half of the MS patients had active, ongoing HHV-6 infections. HHV-6 was also isolated from peripheral blood mononuclear cells (PBMC) from 3/5 MS patients who were in relapse or had progressive disease and was identified as HHV-6 Variant B. These preliminary results support the hypothesis that HHV-6 may be a co-factor in the pathogenesis of some cases of MS.     

Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.     

Cytomegalovirus and human herpesvirus-6 trans-activate the HIV-1 long terminal repeat via multiple response regions in human fetal astrocytes

Cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6) infection stimulated HIV-1 replication and trans-activated the HIV-1 promoter (the long terminal repeat or LTR) to a similar extent in transfected, nonimmortalized, human fetal astrocytes. CMV infection increased basal LTR expression by approximately sevenfold, while HHV-6 infection increased basal LTR expression by fourfold. This enhancing effect required cell-cell contact between CMV-infected or HHV-6-infected and LTR-containing cells. To determine the target regions on the HIV promoter that respond to CMV and HHV-6 trans-activation, several modified LTR-reporter gene constructs were tested. Loss of functional NFkappaB, Sp1, or upstream modulatory sites on the LTR caused significant reduction ofbasal LTR expression in astrocytes. These elements also mediated the trans-activation events during HHV-6 or CMV infection in astrocytes, though to varying degrees. Electrophoretic mobility shift assays (EMSA) indicated that core, enhancer, and upstream modulatory regions of the LTR interacted specifically with nuclear proteins from both uninfected and CMV- or HHV-6-infected human fetal astrocytes. CMV or HHV-6 infection did not appear to induce unique, LTR-specific nuclear binding proteins, but rather enhanced the relative proportion of some of the existing protein complexes, in particular, the complexes formed with the AP-1 binding sites on the HIV-1 LTR (nt - 354 to - 316). Our data suggest that CMV or HHV-6 trans-activation of HIV LTR activity in human fetal astrocytes proceeds via intracellular molecular interactions involving herpesviral gene products, cellular proteins, and multiple sites on the LTR upstream of the TATA box. The pattern of LTR activity in astrocytes suggests that host cell factors modulating HIV expression may differ from those dominant in T-cells or immortalized astroglia, and this could contribute to differences in the astrocyte's ability to support HIV replication.