Ten adult patients with active HHV-6 variant A infections and clinical infectious mononucleosis-like disease (IM) were studied over a period of 32 weeks after onset of disease for their viral DNA load, changes in peripheral blood T-lymphocytes and subpopulations and frequency of cell death in peripheral blood cells. The data were collected as the basis for an advanced computer simulation study for which available data in the literature were too varied. Since the exact time of primary infection of the patients was not known and thus no time relationship of viral effects at cellular level were determined, we supplemented such data from separate tissue culture studies using HHV-6 alpha infection of HSB2 cells. Patients with IM demonstrate an increase in-HHV-6 DNA copies from 0 to 8.2 log 10/5 microL blood within 4 weeks return to normal by 16 weeks. Total T-lymphocytes follow infection with a 20-fold increase above normal peaking at 8-10 weeks and then return to normal by 24-28 weeks. Coincidently, less mature lymphoid cells carrying markers for stem cells, thymic cortical and medullary cells increase 8-10-fold indicating an enhanced mobilization of such cells from premature cell compartments. Cell death in peripheral mononuclear cells peaked with 30% at 8 weeks after onset of clinical disease and normalized by 24 weeks. HHV-6 replication in cell culture as determined by antigen expression, electron microscopy and harvest of infectious virus indicated a complete cycle of virus infection and replication of at least 6 days. The presented data compare well with others from the literature and will serve for testing in a computer simulation model, which is the subject of a forthcoming paper.

Background: HHV-6 is a ubiquitous virus and its infection usually occurs in childhood and then becomes a latent infection. HHV-6 reactivation has been shown to play a role in the pathogenesis of AIDS and several other diseases. Objectives: To determine what role HHV-6 infection or reactivation plays in the pathogenesis of multiple sclerosis (MS) and chronic fatigue syndrome (CFS). Results: Twenty-one MS and 35 CFS patients were studied and followed clinically. In these patients, we measured HHV-6 IgG and IgM antibody levels and also analyzed their peripheral blood mononuclear cells (PBMCs) for the presence of HHV-6, using a short term culture assay. In both MS and CFS patients, we found higher levels of HHV-6 IgM antibody and elevated levels of IgG antibody when compared to healthy controls. Seventy percent of the MS patients studied contained IgM antibodies for HHV-6 late antigens (capsid), while only 15% of the healthy donors (HD) and 20% of the patients with other neurological disorders (OND) had HHV-6 IgM antibodies. Higher frequency of IgM antibody was also detected in CFS patients (57.1%) compared to HD (16%). Moreover, 54% of CFS patients exhibited antibody to HHV-6 early protein (p41/38) compared to only 8.0% of the HD. Elevated IgG antibody titers were detected in both the MS and the CFS patients. PBMCs from MS, CFS and HD were analyzed in a short term culture assay in order to detect HHV-6 antigen expressing cells and to characterize the viral isolates obtained as either Variant A or B. Fifty-four percent of MS patients contained HHV-6 early and late antigen producing cells and 87% of HHV-6 isolates were Variant B. Isolates from CFS, patients were predominately Variant A (70%) and isolates from HD were predominately Variant B (67%). Moreover, one isolate from OND was also Variant B. Persistent HHV-6 infection was found in two CFS patients over a period of 2.5 years and HHV-6 specific cellular immune responses were detected in PBMCs from ten CFS patients. Conclusions: In both MS and CFS patients, we found increased levels of HHV-6 antibody and HHV-6 DNA. A decrease in cellular immune responses was also detected in CFS patients. These data suggest that HHV-6 reactivation plays a role in the pathogenesis of these disorders. (C) 2000 Elsevier Science B.V. All rights reserved.

Because human herpesvirus-8 (HHV-8) DNA has been found in multiple myeloma (MM) patients by polymerase chain reaction, it was suggested that HHV-8 may play a role in the transformation of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore, 362 MGUS sera with and without progression to MM were tested for IgG antibody to HHV-8. Only 7.8% of the MGUS sera contained HHV-8 antibody to lytic proteins, and IgG antibody to HHV-8 latent antigen was even lower than lytic antibody (2.9%). No differences were observed in the distribution of antibody to HHV-8 in sera from MGUS patients who progressed to MM. The seroprevalences of HHV-8 in MGUS (7.8%), MM (5.4%), and healthy donors (5.9%) were similar, thus arguing for the lack of epidemiologic evidence of HHV-8 participation in the pathogenesis of MM. MGUS patients were immune competent in response to Epstein-Barr virus (EBV) infection because 97% contained antibody to EBV virus capsid antigen.

Since the Kaposi's sarcoma-associated herpesvirus (KSHV also referred to as HHV-8, human herpesvirus-8) was discovered it has been shown that the virus is associated with all cases of Kaposi's sarcoma (KS) classical, endemic, or AIDS associated. In the numerous countries where the seroprevalence of this virus has been studied, data demonstrate that the virus is not ubiquitous in general healthy human populations as is the case with other human herpesviruses. Many seroprevalence studies to detect antibodies to HHV-8 have now been conducted using a variety of immunologic techniques. While these assays are not in total agreement and may overstate or understate the positivity of sera in the general population, they all show similar general antibody trends. For general populations the seroprevalence in sub-Saharan Africa is the highest, approximately 40% positive; in Mediterranean countries the seroprevalence is approximately 10%; whereas northern European, southeast Asian, and Caribbean countries have seroprevalence rates in the 2-4% range. In the United States, a 'mixing bowl' country the seroprevalence is in the range of 5-20%. In people with KS whether AIDS associated, classical, or endemic and other HHV-8 associated diseases such as multicentric Castleman's disease and certain body cavity lymphomas (BCL), also called primary effusion lymphoma (PEL) the seroprevalency rates are >90%. In populations with HIV-1 infection but no diagnosis of KS, the seroprevalency rates are elevated (20-50%) above those in the general population except in southeast Asia and the Caribbean where no AIDS associated KS has been reported. No correlation has been found between the presence of KSHV antibodies and other malignancies.

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.

A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1(+) homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.

55 Background: HHV-8 has been implicated in the pathogenesis of Kaposi's sarcoma (KS) as well as body cavity B-cell lymphoma based on PCR analysis of tissues and antibody to viral latent and late proteins. Materials & Methods: We developed an IgG antibody ELISA to HHV-8 using whole viral lysate which contained on average 1.9 × 109 viral particles/ml as well as >1.08 mg/ml of protein. An IFA to HHV-8 lytic antigen was made using the KS-1 cell line. In addition, Western blots to structural virus proteins were developed to elucidate immunodominant proteins that may be detected in sera. Results: Our serological data showed that approximately 11% of the healthy donors in the U.S.A. contained IgG antibody to HHV-8 compared to 12% in Thailand, 4% in India, and 15% in Malaysia (ethnic Chinese, Malays, and Bidayuhians). The antibody titer in healthy donors' sera by IFA ranged 1:20 - 1:320 compared to 1:320 - >1:10,000 in classical KS sera. Similar titers were evident by ELISA, and these assays correlated (p <0.0001, r=0.915). Approximately 50% of HIV-1 (+) homosexuals and >40% HIV-1 (+) IV drug users have HHV-8 serum antibody which suggests being at higher risk for KS. Sera from 93% of KS patients both HIV (+) and HIV (−) contained elevated IgG antibody as detected by IFA and ELISA. Body cavity B-cell lymphoma is the only other disease found associated with increased HHV-8 antibody levels. No evidence of HHV-8 antibody was evident in sera from multiple myeloma, Hodgkin's disease, acute and chronic leukemia, and other B- and T-cell lymphomas from the U.S. population. PCR in situ hybridization for HHV-8 DNA corroborated the disease association results found with the antibody tests. Conclusion: Our data showed variations in infection of HHV-8 in U.S.A. vs. India, Malaysia, and Thailand. Although KS has not been reported in Southeast Asia, HHV-8 infection may be linked to other diseases there. Our ELISA contains a majority of the viral structural proteins and, therefore, detects broad-spectrum antibodies which may be missed by other assays. Our IFA is directed against lytic HHV-8 antigens and correlates well with the ELISA assay. Therefore, these assays may be useful tools in large-scale seroepidemiological studies and clinical follow-up for at-risk individuals. Our findings indicate that the prevalence of HHV-8 is extremely low regardless of the geographic location suggesting that HHV-8 is an important human pathogen associated with KS or other unknown diseases compared to other human gamma, beta, and alpha herpesviruses.

To the Editor: Epidemiologic evidence strongly suggests that young adult Hodgkin's disease (YAHD) is a rare sequelum of a late (adolescence or young adulthood) infection with a common childhood virus, analogous to the appearance of paralysis after natural infection with poliovirus.[1][1] Although

In order to investigate the levels of HHV-6 infection and elevated antibodies to HHV-6 in HIV-1-infected asymptomatic and symptomatic patients, peripheral blood mononuclear cells were (PBMC) cultured. As patients progressed from asymptomatic HIV infection to AIDS, there was a concurrent increase in replicating HHV-6. Plasma obtained from several of these patients showed the presence of IgM antibody and a significantly elevated level of HHV-6 IgG antibody. Serial samples of plasma from 10 AIDS patients collected over a period of 4 years were assayed for the detection of HHV-6 core protein (gp116/64/54) by antigen capture ELISA. The results demonstrated that either a persistent infection or reactivation can occur based on the degree of fluctuation in HHV-6 antigen detected. ELISA to HHV-6 purified viral proteins, i.e., early (p41/38) and late (gp110), demonstrated that IgG antibody to gp110 did not differentiate between HIV-1-infected and healthy donors. IgG and IgM antibody to p41/38, however, showed a significantly higher prevalence in HIV-1-infected individuals (56.7-85.3%) than in normal healthy donors (19.0%), suggesting virus activation. PBMC culture from the AIDS patients expressing significant peaks of HHV-6 core antigen (gp116/64/54) in their plasma showed that in most cases, HHV-6 early and late antigens were detectable; however, those patients with consistently low antigen peaks had no detectable antigens in their PBMC. Only 55% of PBMC cultures established from IgM antibody-positive HIV-1-infected asymptomatic and AIDS patients expressed HHV-6 antigens in the short-term cultures, but HHV-6 antigens could not be demonstrated in PBMC culture from 4 IgM-antibody-positive healthy donors. HHV-6 isolates obtained from the HIV-1-positive patients were predominantly HHV-6 variant A, compared to healthy donors. Based on the data presented here, it is evident that the levels of HHV-6 infection increased in HIV-1-infected asymptomatic individuals as they progressed to AIDS. Our immunovirological data on HHV-6-infected individuals with HIV infection support a role for HHV-6 in the pathogenesis of AIDS. We believe that simultaneous active infection with HIV-1 and HHV-6 may contribute to enhanced immune suppression perhaps leading to disease manifestations.

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.

Background: Human Herpesvirus-8 or KSHV has been implicated in the pathogenesis of Kaposi's sarcoma (KS). HHV-8 has also been associated with body cavity B-cell lymphoma. Sero-epidemiological studies for the prevalence of HHV-8 has been either by IFA using latently infected cells or cells expressing late antigens after chemical treatment. Recombinant capsid proteins or peptides have also been utilized to detect antibodies by ELISA. In order to (a) establish an ELISA for HHV-8 antibody, (b) use the cells expressing late HHV-8 proteins as a substrate for IFA, (c) for molecular characterization of the virus, and (d) for use in infectivity studies, a large amount of HHV-8 was needed. We succeeded in obtaining such amounts from the KS-1 cell line, established from a body cavity lymphoma of an HIV-negative patient by conditioning the cells. Methods and Results: KS-1 cells prior to conditioning did not reveal extra cellular virus; however, after conditioning, herpes virions in all stages of morphogenesis were observed. A 700x preparation of the virus observed with negative staining contained 2 × 109 particles/ml and had a protein concentration of 2.99 mg/ml. Using a lysate prepared from pelleted virus, 22% of normal adult donors, and 33% of non-HIV positive homosexuals without KS had antibody to HHV-8. All 21 sera from classic KS tested and 84% of HIV-positive patients with KS contained IgG antibody to HHV-8. Six out of eight sera from preKS patients were positive. The antibody titer by ELISA of KS patients generally was >1:1280. About 25% of blood donors had a titer of 80-320. By IFA all sera from KS patients tested had a titer range of 1:640 - >1:5120 compared to 1:10 - 1:160 in others, showing a very good correlation between ELISA and IFA. We were able to transmit HHV-8 to human cord blood mononuclear cells. Infection of other suspension and monolayer cells are in progress. The HHV-8 from KS-1 cells was free of EBV, HCMV, HHV-6, HHV-7, HSV, VZV, and HIV. A purified virus preparation analyzed by Western blot recognized viral structural proteins from KS sera. A pilot preparation of HHV-8 had a density of 1.15 g/cm3 on a sucrose gradient. Further molecular studies are in progress. Conclusion: We have succeeded in producing large quantities of infectious HHV-8 from conditioned KS-1 cells, which is an invaluable resource for basic, applied research, and immunodiagnostic development. Our initial data on sero-epidemiology show that in normal populations its prevelance is much lower that other human herpes viruses (EBV, HHV-6, HHV-7, CMV, VZV, HSV-1). In KS patients (classical or other) the prevelance of HHV-8 is significantly higher and antibody titers are highly elevated. Because of higher prevalence of HHV-8 in KS, it may be a major etiological co-factor in its pathogenesis.

Because of the demonstrated presence of Human Herpesvirus-6 (HHV-6) in various tissues and organs of HIV-1 infected AIDS patients, and its implication in the pathogenesis of AIDS, we followed two AIDS patients for active HHV-6 infection for two years. Evidence of active HHV-6 infection was demonstrated by immunovirologic assays in both patients; however, the profile of infection in the two patients varied. One patient showed appearance and disappearance of HHV-6, indicating virus reactivation, whereas the other patient showed chronic or persistent HHV-6 infection. Both patients' peripheral blood mononuclear cells (PBMC) contained HIV-1 when their CD4 cell count was approximately 200 mm3. No active HHV-6 was demonstrated in a healthy donor, who was used as a control for this study.

An HHV-6 antigen capture assay measuring gp116/64/54 antigen was developed. This ELISA is specific for HHV-6 Variants A and B, does not cross react with other human herpesviruses, is sensitive, stable, quantitative, and can detect antigen in body fluids and cell cultures. Relative to virus isolation or techniques for measuring HHV-6 nucleic acids, the assay is much simpler and less expensive to perform. Plasmas/sera (413) obtained from healthy donors, children with Exanthem subitum, febrile illnesses, patients with Chronic Fatigue Syndrome, and AIDS patients tested by antigen capture assay demonstrated that the assay is useful in clinical laboratory settings. The capture assay can also be used to monitor cell cultures for virus isolation, production, quantitation, and antiviral agent screening.

PURPOSE The purpose of this study was to determine whether HIV-1 and HHV-6 are capable of infecting and inducing morphological and ultrastructural changes in corneal epithelial cells in vitro. METHODS Primary and transformed corneal epithelial cell cultures were infected with HIV-1 or HHV-6 in vitro and analyzed for the presence or absence of viral antigens, DNA sequences, viral particles and inclusions. RESULTS HIV-1 antigens were detected in 8% of the HIV-1 infected cells and early HHV-6 antigens were present in 12% of the HHV-6 infected cells. The presence of viral DNA sequences in the cultures confirmed these findings. Cells infected with HIV-1 morphologically were not different from uninfected cells, whereas the morphology of HHV-6 infected cells was very similar to cells infected with other human herpesviruses. Cytoplasmic tubuloreticular inclusions were detectable in corneal epithelia cells infected with HIV-1 and intact viral particles were visible only in PBMC used to recover HIV-1 from these cultures. Viral inclusions were also observed in corneal epithelial cells infected with HHV-6. CONCLUSION These data indicate that HIV-1 and HHV-6 are capable of infecting corneal epithelial cells in vitro, but the viruses are not entering these cells via CD4 or galC receptors. This basic information is important in determining the pathogenic mechanism(s) involved in the development of AIDS-associated corneal disorders.

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 tester 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 HN 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.

PURPOSEThe purpose of this study was to define the agents involved in the development of acquired immune deficiency syndrome (AIDS)-associated retinitis. To achieve this goal, the authors determined the frequency and proximity of the simultaneous presence of human immunodeficiency virus (HIV)-1, human herpesvirus (HHV)-6, and human cytomegalovirus (HCMV) in retinas of patients with AIDS with and without AIDS-associated retinitis.METHODSRetinal sections from 50 globes from patients with AIDS were analyzed for the presence of viral antigens and transcripts. Group 1 contained 13 globes from patients with HCMV infection. Group 2 contained 20 globes from patients with retinal lesions of uncertain etiology in which HCMV antigen and transcripts were not detected. Group 3 contained 17 globes from patients with no retinal lesions.RESULTSRetinal sections from all 13 globes (group 1) were positive for HCMV antigens and HIV-1 antigens and transcripts. Six of the 13 retinas were also positive for HHV-6 antigens and transcripts. Sections from 13 of the 20 globes (group 2) were positive for HIV-1 antigens and transcripts, and 5 of these 13 were also positive for HHV-6 antigens and transcripts. Multiple areas in sections from two of the HIV-1-positive retinas showed coinfection with HHV-6. All 17 globes (group 3) were positive for HIV-1 antigens and transcripts. Ten of these 17 retinas were also positive for HHV-6 antigens. Human cytomegalovirus antigens were not detectable in retinas from groups 2 and 3. No viral antigens or transcripts were detectable in retinal sections from 10 HIV-1 negative donors.CONCLUSIONThe coexistence of HIV-1 and HHV-6 activity in more than 50% of retinas without HCMV infection suggests that HIV-1 and HHV-6 alone or in combination may predispose retinal tissue to other opportunistic agents such as HCMV during the development of AIDS-associated retinitis.