Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders.

A novel human B-lymphotropic virus (HBLV) was isolated from the peripheral blood leukocytes of six individuals: two HTLV-III seropositive patients from the United States (one with AIDS-related lymphoma and one with dermatopathic lymphadenopathy), three HTLV-III seronegative patients from the United States (one with angioimmunoblastic lymphadenopathy, one with cutaneous T-cell lymphoma, and one with immunoblastic lymphoma), and one HTLV-III seronegative patient with acute lymphocytic leukemia from Jamaica. All six isolates were closely related by antigenic analysis, and sera from all six virus-positive patients reacted immunologically with each virus isolate. In contrast, only four sera from 220 randomly selected healthy donors and none from 12 AIDS patients without associated lymphoma were seropositive. The virus selectively infected freshly isolated human B cells and converted them into large, refractile mono- or binucleated cells with nuclear and cytoplasmic inclusion bodies. HBLV is morphologically similar to viruses of the herpesvirus family but is readily distinguishable from the known human and nonhuman primate herpesviruses by host range, in vitro biological effects, and antigenic features.

Ultrastructural characterization of a new human B lymphotropic DNA virus (human herpesvirus 6) isolated from patients with lymphoproliferative disease.

A new DNA virus, designated "human B lymphotropic virus" or "human herpesvirus 6" (HBLV), has been isolated from the peripheral blood leukocytes of patients with various lymphoproliferative disorders, in some instances also associated with human immunodeficiency virus (HIV-1) infection. HBLV, propagated in vitro in human cord blood lymphocytes, was found to be ultrastructurally similar to members of the herpesvirus family. It is an enveloped virus with an icosahedral nucleocapsid made up of 162 capsomeres. Unenveloped nucleocapsids in the cytoplasm wee always coated with a tegument, a feature also described for human cytomegalovirus (HCMV). However, the nucleoprotein core of HBLV does not have the beaded appearance as that of HCMV, nor do HBLV-infected cells contain the skein-like structure. Immune electron microscopy demonstrated the presence of specific antibodies to viral envelope and internal antigens in sera of infected patients, indicating that this virus is a possible human pathogen. These and previously reported characteristics are consistent with the HBLV being a new and unique DNA virus morphologically belonging to the herpesvirus family.

Identification of the virions in the in vitro L1210(V) leukemia cell lines by morphological, virological, and immunological techniques.

In vitro L1210 (V) cell lines contained abundant intracytoplasmic A-particles, numerous C-type particles, a small number of B-type particles, and occasional intracisternal A-particles. The intracytoplasmic A-particles were incorporated into both spiked (B-type) and smooth-surfaced (C-type) particles formed at the budding site. Both B-and C-type particles also developed by gradual accululation of neucleooid material. The particles, particularly the C-type, exhibited a wide range of densities. The cells showed strong surface immunofluoresence for both murine mammary tumor virus and Gross murine leukemia virus antigens and variable degrees of cytoplasmic immunoflurescence for the protein antigens (p1 to p6) of Rauscher leukemia virus. The cells, the culture supernatant, and the purified virus each gave positive reactions with murine mammary tumor virus and murine leukemia virus antisera by immunodiffusion. The viral particles failed to infect C57BL, C57BL/6 X DBA/2F1 (hereafter called BD2F1), BALB/c, Af,and RIIIf mice. Howver, the cells were highly tumorigenic in BD1F-1 mice, moderately tumorigenic in BALB/c mice, but not tumorigenic in C57BL, Af, and RIIIf mice.

HHV-6 inhibition by two polar compounds.

Dimethyl sulphoxide and dimethyl formamide, two polar compounds and powerful cell differentiation inducers, inhibit HHV-6 infection when added to HHV-6-infected HSB2 cultures. This was established by a delay in the time-course of infection and in the development of virus-induced cytopathic effects. Furthermore, viral titration of supernatants showed a significant reduction (3 log10) of the number of infectious particles. Electron microscopy confirmed that viable cells and extracellular virions were present in the cultures containing the polar compounds, while in the non-treated cultures all cells were lysed and no extracellular virus was evident. The mode of action of these compounds is still unclear and warrants further investigation.

Propagation and characterization of human herpesvirus-7 (HHV-7) isolates in a continuous T-lymphoblastoid cell line (SupT1).

After initial culture of HHV-7 in PHA-stimulated human cord blood mononuclear cells (HCBMC), six HHV-7 isolates were propagated successfully in an immature continuous T-lymphoblastoid cell line SupT1. All six isolates infected efficiently the SupT1 cells, and the infected cells became grossly enlarged and multinucleated 7-21 days post-infection. Various stages of HHV-7 morphogenesis were detected. Cell-free supernatants from HHV-7-infected SupT1 cells were infectious to HCBMC as well as to SupT1 cells. The HHV-7-infected SupT1 and HCBMC cell lysates contained more infectious virus than the centrifuged cell culture fluid supernates from the same culture. The HHV-7 isolates H7-2, H7-3, JHC, and JB, concentrated 500 times, had average infectivity titers of 10(3.0) TCID50/ml while strains H7-4 and KHR titered approximately 1-2 logs higher. When all six HHV-7 isolates were propagated in SupT1 and culture fluid supernatants were examined 14-21 days post-infection by negative stain electron microscopy they contained an average of 1.9 x 10(9) virus particles/liter. IFA and ELISA, using HHV-7/SupT1 cell lysate as an antigen, seem to correlate well in detecting high and low HHV-7 antibody in sera from chronic fatigue patients and healthy donors as controls. HHV-7 from SupT1 cell culture was free of HHV-6 and other human herpesviruses as tested by PCR, and the HHV-7 PCR signal was still strong when the viral preparation was diluted to 4.82 x 10(2) genome copies. Since HCBMC are expensive to obtain and available in only small amounts, it is difficult to obtain large quantities of HHV-7 antigen. On the other hand, the SupT1 cell is an excellent source to produce consistently sufficient quantities of HHV-7 for purification studies, development of immunodiagnostics, in vivo infectivity studies, evaluation of antiviral drugs, and molecular biological studies.

Molecular studies of HHV-6.

Methods for the purification of enveloped HHV-6 virions and the viral DNA are presented. The viral genome is estimated to be 170,000 base pairs in size and does not appear to contain inversions due to absence of submolar bands by restriction enzyme analyses. The genomes of two independent HHV-6 isolates, HHV-6GS and HHV-6Z29, showed restriction enzyme site pleomorphism. Large scale purification of enveloped HHV-6 was achieved by continuous flow centrifugation utilizing sucrose gradients, DNAse 1 treatment and banding on 10-30% Dextran T10 gradients. The viral proteins were visualized on high resolution two dimensional polyacrylamide gels and the proteins recognized by serum antibody from patient GS were detected by HR2D Western blot analysis and radioimmunoprecipitation assay. The major antigenic proteins were 200, 120, 80, 72, 30 and 19 kDa.

Human herpesvirus-7 (HHV-7).

HHV-7 first isolated in 1990 from a healthy individual, is a ubiquitous agent. The second independent isolation of HHV-7 from a chronic fatigue syndrome patient was reported in 1992. The seroepidemiology of HHV-7 suggested that its prevalence rate in the U.S.A. population is > 85%; however, in Japan a low prevalence rate has been reported. HHV-7 can be more readily isolated from the saliva than HHV-6. The primary infection of HHV-7 appears later in life than HHV-6. No disease has been reported that is etiologically linked to HHV-7. HHV-7 is more closely related to HHV-6 and the human cytomegalovirus than other members of the human herpesvirus family.

Antiviral activity in vitro of Kutapressin against human herpesvirus-6.

The recently discovered human herpesvirus-6 (HHV-6) is being associated with an increasing number of conditions in which there is evidence of immunologic dysfunction. A number of widely available antiviral agents have shown little or no activity against the virus. We found that Kutapressin (KU), a drug that has been available to practicing physicians for over 50 years, has potent, previously unexpected antiviral effects. Cells known to allow replication of HHV-6 were infected with the virus, under various conditions. Either pretreatment of the cells prior to infection or treatment shortly after infection, inhibited viral replication by > 90%. Indirect evidence suggests that KU may inhibit viral attachment to cellular receptors, and inhibit intracellular maturation of the virus. Given these in vitro findings, and the low frequency of toxicity reported with the use of KU, clinical trials of this drug in patients with evidence of reactivated HHV-6 infection would seem to be warranted.

Ampligen inhibits human herpesvirus-6 in vitro.

The recently discovered human herpesvirus-6 (HHV-6) is being associated with an increasing number of conditions in which there is evidence of immunologic dysfunction. A number of widely available antiviral agents have shown little or no activity against the virus. We found that Ampligen [Poly (1): Poly (C12U), a synthetic, mismatched, double-stranded RNA, has potent, previously unexpected antiviral effects. Cells known to allow replication of HHV-6 were infected with the virus and treated with Ampligen under various conditions. When cells were pretreated with Ampligen (concentrations of 100 or 200 micrograms/ml) prior to infection or treated shortly after infection, viral replication was inhibited by 46-98%. At 100 and 200 micrograms/ml, Ampligen also inhibited the DNA polymerase activity of HHV-6 by 42-98%. When lower concentrations of Ampligen (10 and 50 micrograms/ml) were used, only pretreatment of cells, with Ampligen, followed by virus infection and carrying the infected cells with Ampligen, significantly inhibited HHV-6 infection (83.7 and 89.1% respectively). Indirect evidence suggests that Ampligen may inhibit viral attachment to cellular receptors and/or inhibit intracellular maturation of the virus. The above concentrations of Ampligen were not toxic to the cells used in the study. Given these in vitro findings, and the low frequency of toxicity reported with the use of Ampligen, clinical trials of this drug in patients with evidence of reactivated HHV-6 infection would seem to be warranted.

Concurrent replication of a papovavirus and a C-type virus in the CCL 33 porcine cell line.

A papovavirus, CCL 33 PV, isolated from a porcine cell line, CCL 33 (GT), was characterized. Based on a comparison of four isoenzyme systems, CCL 33 (GT) and CCL 33 (ATCC), obtained directly from the American Type Culture Collection, appeared to be the same. In addition to the previously characterized C-type virus of CCL 33 cultures, CCL 33 (GT) produced CCL 33 PV in high quantity, but CCL 33 (ATCC) produces a papovalike virus, presumably the same as CCL 33 PV, in barely detectable amounts. Serological results showed that CCL 33 PV is apparently identical to a papovavirus (SPV) isolated by Newman and Smith after inoculation of CCL 33 with concentrated porcine trypsin. These studies extend the characterization of this papovavirus, demonstrating that CCL 33 PV is weakly hemagglutinating after neuraminidase treatment, has a high affinity for a component of fetal bovine serum and is highly infectious in appropriate porcine cell systems rather than very defective as reported previously. Moreover, it was concluded from the data that CCL 33 PV is probably indigenous to the CCL 33 porcine cell line.

Ultrastructural comparison of a virus from a Rhesus-monkey mammary carcinoma with four oncogenic RNA viruses.

The ultrastructure and morphogenesis of Mason-Pfizer monkey virus, isolated from a mammary carcinoma in a Rhesus monkey, was compared with those of murine mammary tumor virus, murine leukemia virus, L1210 leukemia-associated virus, and avian myeloblastosis virus. The simian virus resembled murine mammary tumor virus and the L1210 virus in that it produced intracytoplasmic particles that were enveloped during budding. It resembled L1210 virus and murine leukemia virus in budding with smooth envelopes. It differed from all the others in being more fragile. These similarities, combined with biochemical characteristics reported elsewhere in this issue, suggest that the monkey virus is an oncogenic RNA virus.

Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell.

Chinese hamster ovary cells harbour intracytoplasmic virus-like particles of type A which are closely associated with sites of microtubule formation. We report here the enhanced proliferation of these particles and their release at the cell membrane by using either 5-bromodeoxyuridine or dibutyryl cyclic AMP. The extracellular mature particles are similar in morphology to retroviruses of type B. Close association of the type A virus precursors with microtubule organizing centres, i.e. kinetochores, centrioles and basal bodies, and with microtubules per se, is confirmed by studying the effects of the microtubule inhibitors Colcemid and vincristine sulphate. The role of microtubules in the activation and transport of the intracytoplasmic type A particles is discussed.

Human herpesvirus-7 (HHV-7): current status.

BACKGROUND: Human herpesvirus-7 (HHV-7) is a newly discovered virus and very little is known about its prevalence, biologic, immunologic and molecular biology aspect. Besides the HHV-7 etiologic role in a few cases of exanthem subitum, its association with other diseases has not been reported. OBJECTIVES: To review what is currently known about HHV-7. RESULTS: HHV-7 was first isolated in 1990 from purified T-cells from a healthy individual. Following this report, an independent isolation of HHV-7 was reported from the mononuclear cells (PBMC) of a chronic fatigue syndrome patient. HHV-7 is closely related to human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV), but is distinct from Epstein-Barr virus (EBV), herpes simplex virus and varicella zoster virus. Using polyvalent and monoclonal antibodies, several HHV-7 viral proteins were identified, ranging from 136 to 30 kDa. HHV-7 infection occurs later than HHV-6, which appears in early childhood. HHV-7 is ubiquitous, and its prevalence rate is >85% in the US population, although its rates of prevalence in Japan is lower than in the USA and Europe. HHV-7 is frequently isolated from saliva; however, HHV-7 has been consistently isolated from PBMC from young children as well. Several cases of exanthem subitum have been linked to primary infection of HHV-7, suggesting that it may also cause exanthem subitum. Primary infection with HHV-7 was also reported from a patient with features of hepatitis and exanthem subitum. This virus was also isolated from tissues from a case of hepatosplenomegaly and pancytopenia lacking either EBV or HCMV. Thus far, no other disease associated with HHV-7 has been reported. Only one continuous T-cell line (SupT1) can support the replication of HHV-7, but the virus yield is extremely low. CONCLUSIONS: It has been about 4 years since this member of the human herpesvirus family was reported. In the coming years, more data will be available on the epidemiology, biology, immunology, molecular biology, and pathogenesis of HHV-7. The finding of reciprocal interference between HHV-7 and HIV-1, suggesting competition at the receptor level is important, needs further work and here HHV-7 may play a role as a negative cofactor in the natural history of HIV infection. Because of HHV-7 interaction with HIV-1, the possibility of its vertical transmission needs to be investigated. This review on HHV-7 is intended to provide current information on HHV-7.

Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus-6 isolates.

Fifteen human herpesvirus-6 (HHV-6) isolates from normal donors and patients with AIDS, systemic lupus erythematosis, chronic fatigue syndrome, collagen-vascular disease, leukopenia, bone marrow transplants, Exanthem subitum (roseola), and atypical polyclonal lymphoproliferation were studied for their tropism to fresh human cord blood mononuclear cells, growth in continuous T cell lines, reactivity to monoclonal antibodies, and by restriction enzyme banding patterns. All isolates replicated efficiently in human cord blood mononuclear cells, but mitogen stimulation of the cells prior to infection was required. The ability to infect continuous T-cell lines varied with the isolates. Isolates similar to GS prototype infected HSB2 and Sup T1 cells and did not infect Molt-3 cells, whereas isolates similar to Z-29 infected Molt-3 cells but not HSB2 and Sup T1 cells. Some of the monoclonal antibodies directed against the HHV-6 (GS) isolate showed reactivity with all isolates tested, but others only reacted with HHV-6 isolates similar to the GS isolate and not with those similar to Z-29 isolate. Restriction enzyme analysis using EcoRI, BamHI, and HindIII revealed that HHV-6 isolates from roseola, bone marrow transplant, leukopenia, and an HIV-1-positive AIDS patient from Zaire (Z-29) were closely related but distinct from GS type HHV-6 isolates. Based on the above findings, we propose that, like herpes simplex virus types 1 and 2, the 15 HHV-6 isolates analyzed can be divided into group A (GS type) and group B (Z-29 type).

Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).

Details of the productive infection of established human cell lines of diverse origin by HBLV (also designated Human Herpesvirus 6) are described in this report. The infection and replication of HBLV in several T and B lymphoid and other cell lines was observed by electron microscopic examination, by the detection of viral antigen expression by indirect immunofluorescence assay (IFA) and by the presence of HBLV DNA by Southern blot hybridization. Several of these cell lines produced large amounts of virus. For this reason and because of the absence of other human herpesviruses, these lines have provided a valuable resource for the preparation of reagents and the development of assays for the detection and characterization of HBLV. The isolation and characterization of new HBLV isolates from patients with chronic fatigue syndrome were also facilitated by using some of the cell lines reported here. The host range of HBLV in established cell lines, therefore, does not appear to be limited to the B lymphocytes, as initially suggested by in vivo studies. The infection of T and B lymphocytes, megakaryocytes and neuronal cells in vitro suggests a need for the evaluation of diverse hematological and neurological disorders to shed light on a possible HBLV involvement.