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Suppressive effects of human herpesvirus-6 on thrombopoietin-inducible megakaryocytic colony formation in vitro

Two clinical observations, the association of human herpesvirus-6 (HHV-6) with delayed engraftment after stem cell transplantation and thrombocytopenia concomitant with exanthema subitum, prompted us to evaluate the suppressive effects of HHV-6 on thrombopoiesis in vitro. Different culture conditions for thrombopoietin (TPO)-inducible colonies in semi-solid matrices were examined. Using cord blood mononuclear cells as the source of haematopoietic progenitors, two types of colonies, megakaryocyte colony-forming units (CFU-Meg) and non-CFU-Meg colonies, were established. The former colonies were identified by the presence of cells with translucent cytoplasm and highly refractile cell membrane, most of which were positive for the CD41 antigen. Although the plating efficiency of both types was much higher under serum-containing conditions than under serum-free conditions, the proportion of CFU-Meg to non-CFU-Meg colonies was consistently higher under serum-free conditions. The plating efficiency of CFU-Meg colonies was doubled by adding stem cell factor to the serum-free matrix. The effects of two variants of HHV-6 (HHV-6A and 6B) and human herpesvirus-7 (HHV-7) on TPO-inducible colonies were then compared. HHV-6B inhibited both CFU-Meg and non-CFU-Meg colony formation under serum-free and serum-containing conditions. HHV-6A had similar inhibitory effects. In contrast, HHV-7 had no effect on TPO-inducible colony formation. Heat-inactivation and ultra-filtration of the virus sample completely abolished the suppressive effect. After infection of CD34(+) cells with HHV-6, the viral genome was consistently detected by in situ hybridization. These data suggest that the direct effect of HHV-6 on haematopoietic progenitors is one of the major causes of the suppression of thrombopoiesis.

Monitoring of human herpesvirus‐6 and ‐7 genomes in saliva samples of healthy adults by competitive quantitative PCR

Human herpesviruses‐6 and ‐7 (HHV‐6 and HHV‐7) are thought to be transmitted during early infancy through saliva. However, the kinetics of the virus shedding in saliva of healthy adults, from whom children are assumed to acquire the viruses, is mostly unknown. This study was conducted to determine how many copies of the genome are secreted in saliva of healthy adults and to clarify the relationship between viral DNA load and virus isolation of HHV‐6 and HHV‐7. Competitive PCR was performed using primer sets in the U42 gene of each viral genome. In saliva samples from 29 healthy adults, HHV‐6 and HHV‐7 DNA was detected in 41.4% and 89.7%, respectively. The average copy number of the HHV‐7 genome in the positive samples was higher than that of the HHV‐6 genome. Follow‐up studies of six seropositive individuals for 3 months showed that the amount of HHV‐7 DNA was constant in each individual and that “high producers” and “low producers” could be distinguished. By contrast, the amount of HHV‐6 DNA varied drastically over time in each individual. Although HHV‐6 was never isolated from the saliva of any of the six individuals during the follow‐up period, HHV‐7 was isolated from each individual several times. The amount of HHV‐7 DNA tended to be higher at the times when the virus was isolated than at the times when the virus was not isolated. These data demonstrate a striking contrast between HHV‐6 and HHV‐7 in the kinetics of genome and virus shedding. J. Med. Virol. 61:208–213, 2000.

Electron Microscopic Study of a Herpes-Type Virus Isolated from an Infant with Exanthem Subitum

Human lymphocytes and MT4 cells infected with a virus isolated from a patient with exanthem subitum were examined by transmission and scanning electron microscopy. The most striking characteristic of the ultrastructure of this herpes‐type virus was that nucleocapsids located outside the nucleus were each coated distinctly with a tegument of moderate electron density. Tubular structures formed due to some mistakes in the viral assembly were also detected in the nucleus. Morphological differentiation of this virus from the other human herpesviruses was discussed. From these observations it was concluded that this virus has the same ultrastructural characteristics as HBLV (HHV‐6).

A morphologically recognizable marker for scanning immunoelectron microscopy: I. T4-bacteriophage

Abstract A simple but useful immunological labeling method for scanning electron microscopy is described in the present paper. Bacteriophage T 4 was suitable as a marker for detecting surface antigens. A method using IgG conjugated to bacteriophage by glutaraldehyde was used successfully with serological specificity for labeling the surface antigens on human red blood cells.

Replication of Human Herpesvirus 6 (HHV-6): Morphological Aspects

Human herpesvirus 6 (HHV-6) was first isolated by Salahuddin et al. (1) from the peripheral-blood leukocytes of six patients with associated lymphoproliferative disorders, and subsequently by Lopez et al. (2) and other research groups (3, 4) from individuals seronegative as well as individuals seropositive for human immunodeficiency virus (HIV). Yamanishi et al. (5) probably isolated a different strain of the same virus from the peripheral-blood lymphocytes of patients with exanthem subitum.

In vitro cytopathology and pathogenicity to inbred mice shown by five variants of a laboratory strain of type 1 herpes simplex virus

SummaryThein vitro cytopathology and the neurovirulence to inbred mice demonstrated by five variants originally derived from one laboratory strain (Miyama) of type I herpes simplex virus (HSV-1) were studied comparatively. Three of the variants are syncytial [+GC (LPV), +GC (SPV), +GC (81)] and two are non-syncytial [−GCr and −GCf]. The size of plaques produced by the five variants was found to be in the order of +GC (LPV)>+GC (81)>+GC (SPV)>−GCf>−GCr. The pathogenicity of these variants was compared in three kinds of inbred mice (AKR, C 3 H/He and C 57 BL) after intraperitoneal (IP) or intracerebral (IC) inoculation. The +GC (LPV) variant was the most virulent as shown by the highest mortality of mice by either route of inoculation. The other four variants caused death of mice only after IC inoculation, and among these variants, +GC (81) was shown to be the most virulent. These data indicate that so far as these five variants of the Miyama strain of HSV-1 are concerned, neurovirulence is positively correlated with their cell fusion activity or the size of plaques which they produce. Pre-IP-inoculation with any of the less virulent variants [−GCr, +GC (SPV) and +GC (81)] protected mice from subsequent lethal infection with +GC (LPV) by the same route of inoculation.

Effect of recombinant mouse interferon-β on acute and latent herpes simplex infection in mice

SummaryThe antiviral effect of recombinant mouse interferon-β(rMuIFN-β) on herpes simplex virus type 1 (HSV-1) in experimentally infected mice was examined at several stages of infection as a model for the treatment of human HSV infection. Recombinant MuIFN-β protected mice from lethal intraperitoneal challenge with virulent HSV-1 strains. The in vitro reactivation of HSV from latently infected trigeminal ganglia was also suppressed by treatment with rMuIFN-β. Thus, rMuIFN-β was effective against HSV-1 during acute infection and during in vitro reactivation of latent HSV. However, rMuIFN-β was not effective in preventing the establishment of latent infection, or in eliminating a previously established latent infection.

Mechanism of Differences in Pathogenicity between Two Variants of a Laboratory Strain of Herpes Simplex Virus Type 1

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV‐1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the ‐GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated ‐GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV‐1 was shown to be age‐dependent.

Infection of a human retinal pigment epithelial cell line with human herpesvirus 6 variant A.

A retinal pigment epithelial (RPE) cell line (K‐1034) was examined for its susceptibility to human herpesvirus 6 variant A (HHV‐6A). Exposure of K‐1034 cells to HHV‐6A induced the formation of multinucleated giant cells, which was suppressed by an inhibitor of viral DNA synthesis. In the giant cells, herpesvirus nucleocapsids were demonstrated by electron microscopy and the viral glycoprotein B was detected by immunofluorescence assay. These results indicate that K‐1034 cells are susceptible to HHV‐6A and suggest that HHV‐6A has an ability to directly destroy epithelial cells. J. Med. Virol. 53:105–110, 1997.

Characterization of 17 human immunodeficiency virus-1 carrier cell lines with T cell, myelomonocyte, or megakaryocyte lineages

Abstract Of 29 hematopoietic cell lines tested for susceptibility to human immunodeficiency virus (HIV)-1HTLV-IIIB infection, all CD4+ cell lines became infected. Continuous culturing of infected cell lines resulted in nine HIV-1 carrier cell lines, including, for the first time, an HIV-1 carrier megakaryoblastic cell line, MEG-01/HIV. The immunophenotypic profiles of a total of 17 HIV-1 carrier cell lines (nine newly and eight previously established cell lines) were compared with their respective parental noninfected cell lines. Except for total absence of CD4 expression, the expression of other antigens was variable among the 17 HIV-1 carrier cell lines. Persistent and consistent replication of infectious HIV-1 was detected in all of them in varying quantities. The great variability observed in both the altered marker expression, with respect to that of the noninfected parental cell lines, and in the quantities of persistently produced infectious HIV-1 was, nevertheless, specific to the individual cell lines. Furthermore, the present study demonstrates that there is no apparent correlation in the quantity of HIV-1 produced to either T cell, myelomonocytic cell, or megakaryocytic cell types. Instead, the results suggest that a particular interaction between HIV-1 and individual clonal cell lines may provide insight into the extremely complex immune dysregulation associated with the pathogenesis of acquired immune deficiency syndrome. Thus, the 17 HIV-1 carrier cell lines of diverse origin presented here provide valuable and unique models for further understanding acquired immune deficiency syndrome pathogenesis at the cellular and molecular levels. [P.S.E.B.M. 1993, Vol 202]