Human Herpesvirus 8 Sequences Research Articles

The role of HHV-6 and HHV-7 infections in the development of fibromyalgia

Human herpes virus-6 (HHV-6) and human herpes virus-7 (HHV-7) are immunomodulating viruses potentially affecting the nervous system. We evaluated the influence of HHV-6 and HHV-7 infections on fibromyalgia (FM) clinical course. Forty-three FM patients and 50 control group participants were enrolled. 39.50% (n = 17) FM patients had light A delta and C nerve fiber damage, 27.91% (n = 12) had severe A delta and C nerve fiber damage. 67.44% (n = 29) FM patients had loss of warm sensation in feet, loss of heat pain sensation, and increased cold pain sensation (34.90%, n = 15 in both findings). HHV-6 and HHV-7 genomic sequences in peripheral blood DNA in 23/43 (51.00%) and 34/43 (75.50%) of samples from FM patients and in 3/50 (6.00%) and 26/50 (52.00%) of samples from the control group individuals were detected. Active HHV-6 (plasma viremia) or HHV-7 infection was revealed only in FM patients (4/23, 17.40% and 4/34, 11.80%, respectively). A statistically significant moderate positive correlation was found between A delta and C nerve fiber damage severity and HHV-6 infection (p < 0.01, r = 0.410). 23/43 patients from the FM group and control group participants HHV-6 and 34/45 HHV-7 did have infection markers. A statistically significant moderate positive correlation was found between A delta and C nerve fiber damage severity and HHV-6 infection (p < 0.01, r = 0.410). No difference was found between detection frequency of persistent HHV-6 and HHV-7 infection between FM patients and the control group. Statistically significant correlation was observed between quantitation of changes in QST thermal modalities and HHV-6 infection. There was no correlation between A delta and C nerve fiber damage and HHV-7 infection.

Identification of Viruses in Cases of Pediatric Acute Encephalitis and Encephalopathy Using Next-Generation Sequencing.

Acute encephalitis/encephalopathy is a severe neurological syndrome that is occasionally associated with viral infection. Comprehensive virus detection assays are desirable because viral pathogens have not been identified in many cases. We evaluated the utility of next-generation sequencing (NGS) for detecting viruses in clinical samples of encephalitis/encephalopathy patients. We first determined the sensitivity and quantitative performance of NGS by comparing the NGS-determined number of sequences of human herpesvirus-6 (HHV-6) in clinical serum samples with the HHV-6 load measured using real-time PCR. HHV-6 was measured as it occasionally causes neurologic disorders in children. The sensitivity of NGS for detection of HHV-6 sequences was equivalent to that of real-time PCR, and the number of HHV-6 reads was significantly correlated with HHV-6 load. Next, we investigated the ability of NGS to detect viral sequences in 18 pediatric patients with acute encephalitis/encephalopathy of unknown etiology. A large number of Coxsackievirus A9 and mumps viral sequences were detected in the cerebrospinal fluid of 2 and 1 patients, respectively. In addition, Torque teno virus and Pepper mild mottle viral sequences were detected in the sera of one patient each. These data indicate that NGS is useful for detection of causative viruses in patients with pediatric encephalitis/encephalopathy.

Oral Human Papillomavirus and Human Herpesvirus-8 Infections Among Human Immunodeficiency Virus Type 1–Infected Men and Women in Italy

Oral human papillomavirus (HPV) and human herpesvirus-8 (HHV8) infections are sexually transmitted and respectively associated with the development of oropharyngeal carcinoma and Kaposi sarcoma. The aim of the study was to evaluate HPV prevalence and its possible correlation with HHV8 oral shedding, in relation to sex, human immunodeficiency virus (HIV) behavioral risk factor, and immune function. The study population comprised 100 HIV-infected individuals divided into 3 groups: (1) 38 men who have sex with men (MSM), (2) 24 heterosexual men, and (3) 38 women. DNA was obtained from cells of unstimulated whole saliva. Human papillomavirus sequences were searched for by polymerase chain reaction (PCR) with MY09/MY11 primers or by nested PCR with GP5+/GP6+ primers as the second step. Typing was accomplished by restriction fragment length polymorphism analysis or by direct sequencing or by reverse line blot. Human herpesvirus-8 sequences were detected and quantified by nested PCR and real-time PCR, respectively. Oral HPV infection was present in 37 (prevalence, 37%) of 100 (13 with high-risk and 24 with low-risk types) patients; the most frequent types were HPV16, HPV6, HPV10, HPV61, HPV66, and HPV83. Human herpesvirus-8 DNA was detected in 46 (46%) of 100 subjects. Both infections had the highest prevalence among MSM and the lowest among women; women had a lower prevalence of high-risk HPV types than did both male groups (P = 0.05). An inverse correlation was observed with concomitant oral HHV8 infection (P = 0.007). High prevalence of oral HPV and HHV8 infections was observed; MSM had the highest figures, despite better control of HIV infection.

Chromosomally-integrated human herpesvirus 6 in familial glioma etiology

Human herpesvirus 6 (HHV-6) is a highly neurotropic beta-herpesvirus with demonstrated transformative properties. HHV-6 infection has been implicated in the etiologies of cancers, including lymphoma and leukemia; conditions with brain involvement, including epilepsy and encephalitis; and other disorders. HHV-6 is also the only human herpesvirus that has been proven to integrate into the chromosomes of a proportion (1–12%) of infected individuals. Because several traditional genetic association studies have failed to identify a variant that can account for the established relationship between family history and glioma risk, the possibility that chromosomally-integrated HHV-6 (CI-HHV-6), as a heritable factor, may explain a proportion of familial glioma cases warrants evaluation. To test this hypothesis, the prevalence of CI-HHV-6 in familial glioma cases and related and unrelated cancer-free control groups should be compared. Among glioma-affected families, the inheritance pattern of CI-HHV-6 could be evaluated by constructing pedigrees. If CI-HHV-6 is found to be associated with familial glioma risk, this knowledge could potentially lead to the future development of novel therapeutic and preventive approaches, including vaccines and immunotherapies targeted at the HHV-6 sequences.

Human herpesvirus type 8 variants circulating in Europe, Africa and North America in classic, endemic and epidemic Kaposi's sarcoma lesions during pre-AIDS and AIDS era

Human herpesvirus-8 (HHV-8) variants have been found heterogeneously distributed among human populations living in diverse geographic regions, but their differential pathogenicity in Kaposi's sarcoma development remains controversial. In the present study, HHV-8 variant distribution has been analyzed in classic, iatrogenic, endemic as well as epidemic Kaposi's sarcoma (KS) during pre-AIDS and AIDS period (1971-2008) in countries with different KS incidence rate. DNA samples from cutaneous KS lesions of 68 patients living in Africa (n=23, Cameroon, Kenya and Uganda), Europe (n=34, Greece and Italy) and North America (n=11) have been subjected to PCR amplification of HHV-8 ORF 26, T0.7, K1 and K14.1/15, followed by direct nucleotide sequencing and phylogenetic analysis. Among the 23 African samples, the majority of HHV-8 ORF 26 variants clustered with the subtype R (n=12) and B (n=5). Conversely, the viral sequences obtained from 45 European and North European tumors belonged mainly to subtype A/C (n=36). In general, HHV-8 and K1 variant clustering paralleled that of ORF 26 and T0.7. Genotyping of the K14.1/15 loci revealed a large predominance of P subtype in all tumors. In conclusion, comparison of the HHV-8 sequences from classic or endemic versus AIDS-associated KS showed a strong linkage of the HHV-8 variants with specific populations, which has not changed during AIDS epidemic.

Association of Human Herpesvirus 6 and 8 in Relapsing Remitting Multiple Sclerosis Type during Exacerbation

Multiple Sclerosis (MS) is a chronic demyelinating disease of the CNS with assumed autoimmune etiology. Human herpes viruses have probable effects on relapsing-remitting MS pathogenesis presumably through molecular mimicry and/or bystander mechanisms. In this study we probed the possible contribution of the two herpes viruses, human herpesvirus 6 (HHV-6) and human herpesvirus 8 (HHV-8), in clinically definite multiple sclerosis (CDMS) pa-tients-relapsing remitting type (RRMS) during clinical exacerbations. All patients had no history of immune modulating or suppressing drugs intake in the last 6 months. The peripheral blood samples, from CDMS patients (n = 20) (13F/7M, age (y) = 30.3 ± 3.21) and other immune mediated neurological disorders (OIND) (11F/9M, age = 25.2 ± 12.1), (My-asthenia Gravis, Guillain Barré Syndrome, ischemic stroke in adolescent and young adult with no clear risk factors), as a control group, had been enrolled within 15 months (January-2007-- March -2008). We investigated the existence of specific deoxyribonucleic acid (DNA) sequences belonging to HHV-6 and HHV-8, using polymerase chain reaction (PCR) in the isolated peripheral blood mononuclear cells (PBMCs) and in plasma. PCR demonstrated HHV-6 DNA in 7 cases (35%), HHV-8 sequences in only one cases (5%) in PBMCs from 20 relapsed CDMS patients; all HHV-6 posi-tive cases showed positive plasma results, while the blood samples from 20 OIND patients showed negative results ex-cept one case (5%) out of 9 cases of GBS was positive for HHV-8 in PBMCs. We consequently concluded that there is considerable evidence in this study that proposed the roles of HHV-6 and HHV-8 in MS pathogenesis and clinical ex-acerbation.

Absence of Human Herpesvirus 8 DNA Sequences in Lung Biopsies from Israeli Patients with Pulmonary Arterial Hypertension

Background:Pulmonary hypertension is a severe pulmonary vascular disease leading to rapid deterioration and death. Histological and clinical evidence suggests that smooth muscle proliferation is part of the pathogenesis of the disease. Human herpesvirus 8 (HHV-8) is a γ-herpesvirus that is implicated in malignancies and in Kaposi’s sarcoma. Recently, the association of HHV-8 with idiopathic pulmonary arterial hypertension (PAH) has been found. Objective: The aim of this study was to investigate the presence of HHV-8 in the lung tissue of Israeli patients with PAH. Method: The presence of HHV-8 sequences was investigated by polymerase chain reaction examination in 6 biopsies of patients with pulmonary hypertension. Three patients had idiopathic pulmonary hypertension, 2 patients pulmonary venoocclusive disease, and 1 patient pulmonary hypertension associated with mixed connective tissue disease. Result: We did not find any association between HHV-8 and PAH in these Israeli patients, as all the samples were negative for polymerase chain reaction. Conclusion: Our findings, together with the epidemiological data of HHV-8 prevalence and incidence rates of Kaposi’s sarcoma and PAH in Israel, provide further evidence which argues against an association between HHV-8 infection and PAH.

Serological and molecular detection of HHV-8 in Brazilian populations

Little is known about human herpesvirus 8 (HHV-8) distribution in Brazil. We used indirect immunofluorescence serological assays to determine HHV-8 seroprevalence in two Amerindian tribes from the Amazon region, and blood donors and Kaposi’s sarcoma (KS) patients from the Campinas/SP (Southeastern region). Anti-HHV-8 antibodies were detected in 56.8% of the Amerindians (558/982), in all ages (0-81 years old) and both sexes. In these populations, high prevalence in children younger than 2 years old (44.4%) and children from 2 to 9 years old (35.0%) suggests non-sexual routes of HHV-8 transmission, through vertical transmission or contact to contaminated secretions. HHV-8 seroprevalence in blood donors from Campinas/SP was low (2.8%) and all positive cases were male (9/319) in the fourth and fifth decades of life. Curiously, these individuals were negative to routine serological tests applied in blood banks. Every KS patient assessed in our study was male, with average age of 37 years (27 to 79 years) and anti-HHV-8 positive assays in all cases. In order to determine HHV-8 molecular prevalence, we analyzed DNA from three Amerindian tribes from the Amazon region, KS patients from Campinas/SP and HIV patients from Salvador/BA (Northeastern region). We used Nested-PCR to amplify HHV-8 ORF-26 region by molecular screening. Every ORF-26 amplified sample was also amplified to hypervariable ORF-K1 region for HHV-8 genotyping. We analyzed 384 DNA samples from Amerindian tribes and detected HHV-8 sequences in 3.8% (13/384). KS patients had all DNA samples from skin biopsies and 45.5% from peripheral blood (PBMC) amplified. DNA samples from 148 HIV positive patients were analyzed and HHV-8 sequences were detected in 4% of cases (6/148). Almost all positive DNA samples were amplified to ORF-K1 and determined HHV-8 subtypes. Molecular techniques for amplification and sequencing of two fragments (VR1 and VR2) from ORF-K1 region made possible to build up phylogenetic trees and determine HHV-8 main viral subtypes (A, B, C, D and E) and its variants. Patients with KS from Campinas had subtypes A, B and C detected, with greater frequency of subtype C. Subtypes A and E were detected in Amazon Amerindians. This study is the first to perform genotyping in samples of HIV positive patients from Salvador, detecting subtype B and an unclassified subtype. Thus, it was possible to determine that HHV-8 subtypes A, B, C and E are present in Brazilian populations. As Brazil is a large country with variable population, culture and different geographical characteristics, more HHV-8 epidemiological studies are necessary to establish possible regional differences. This study was supported by grants from the Conselho Nacional de Pesquisa (CNPq) and Fundacao de Apoio a Pesquisa na Bahia (FAPESB).

Human herpesvirus 8 (HHV8) sequence variations in HHV8 related tumours in Okinawa, a subtropical island in southern Japan

Background: Although rare in mainland Japan, classic Kaposi’s sarcoma (KS) is frequently reported in Okinawa, a subtropical island in southern Japan. Human herpesvirus 8 (HHV8) has been identified in the...

Antigenic Open Reading Frames from HHV-8 are Present in Multiple Myeloma Patients and Normal Individuals at Similar Frequency

It has been proposed that the absence of a humoral response to human herpes virus 8 (HHV-8) in patients with multiple myeloma (MM) reflects strain variation or the mutation, or absence, of the antigenic regions of HHV-8 recognized in ELISA screening tests. We therefore assessed DNA sequence of three antigenic regions (ORF65, ORF73 and ORFK8.1) and the transforming hypervariable K1 ORF of HHV-8 in fresh bone marrow cells, bone marrow derived dendritic cells (DCs) and bone marrow stromal cells (BMSCs) from 12 patients with MM and 8 normal individuals. HHV-8 ORFs were detectable by nested PCR in MM patients (ORF65: 67% ORF73: 22% and K8.1: 58%), but were also surprisingly frequent in normal individuals (ORF65: 37%, ORF73: 12.5% and K8.1: 62%). HHV-8 sequences were more frequently detected in cells from BMSC and DC culture than from fresh bone marrow in MM. In contrast no HHV-8 sequences were detected in BMSC from normal individuals. Sequence analysis of ORF65 failed to demonstrate productive mutations in any MM sample. K1 genomic sequences were detected in 42% of MM and 37% of normals and exhibited 98% homology with the K1-A1 HHV-8 strain. In conclusion, our data do not support the presence of a K1-C3 strain of HHV-8 with ORF65 expression deficiency in MM patients. HHV-8 infection appears to be common in the general population when sensitive PCR is employed and multiple samples are analyzed.

A search for human herpesvirus 8(HHV-8) in HIV-1 infected mothers and their infants does not suggest vertical transmission of HHV-8

A search for human herpesvirus 8(HHV-8) in HIV-1 infected mothers and their infants does not suggest vertical transmission of HHV-8

Human herpesvirus-8 in lymphomatous and nonlymphomatous body cavity effusions developing in Kaposi's sarcoma and multicentric Castleman's disease.

Human herpesvirus-8 (HHV-8) has been associated with Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma. Kaposi's sarcoma and multicentric Castleman's disease patients may develop body cavity effusions that, unlike primary effusion lymphoma, are poorly characterized. To better define these effusions, pleural and peritoneal fluids derived from 12 human immunodeficiency virus-seropositive and one seronegative patients affected by Kaposi's sarcoma or multicentric Castleman's disease were analyzed by a combination of morphologic, immunophenotypic, and DNA analyses, including polymerase chain reaction amplification of HHV-8, Epstein-Barr virus, and immunoglobulin heavy-chain (IgH) gene sequences. In addition, HHV-8 serologic status was assessed by using an immunofluorescence assay. All patients were adult men with high antibody titers to HHV-8; 11 of the 13 patients were homosexual/bisexual. Effusions revealed monocyte/macrophage-rich infiltration (10 patients) or large-cell lymphoma with CD45(+)/non-T/non-B phenotype (three of 13 patients); polymerase chain reaction analysis showed the presence of HHV-8 sequences (nine of 13 patients), germline IgH (seven of 12 patients) or clonal IgH rearrangements (four of 12 patients), and rarely Epstein-Barr virus sequences (two of 12 patients). In the setting of HHV-8 infection, two effusion types may occur. One fulfills the criteria for HHV-8-positive PEL (lymphoma-morphology, HHV-8-DNA(+), IgH rearrangement). The other seems more reminiscent of an HHV-8-associated nonneoplastic process (monocyte-macrophage morphology, HHV-8-DNA(+/-), germline IgH). Interestingly, a single case of the latter effusion type harbored a B-cell monoclonal proliferation, which suggests the hypothesis that a prelymphomatous effusion may precede overt body cavity lymphoma.

The human herpesvirus-8 ORF 57 gene and its properties.

Human herpesvirus-8 (HHV-8) is a gamma(2) lymphotropic herpesvirus associated with Kaposi's sarcoma, a major neoplasm of AIDS patients, and with other AIDS-related neoplasms. The HHV-8 ORF 57 gene is conserved throughout the herpesvirus family and has a herpes simplex virus type 1 homologue, IE63 (also termed ICP27), which is an essential regulatory protein and acts at both transcriptional and post-transcriptional levels. We show that, contrary to the published HHV-8 sequence, which predicts a protein of 275 amino acids, the ORF 57 gene is spliced, contains a single intron and encodes a protein of 455 amino acids. For several gammaherpesviruses examined, the upstream coding exon is 16-17 amino acids in length and is rich in methionine residues. When ORF 57 was fused to the gene for enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the cellular splicing factor SC-35. Unlike the IE63-EGFP fusion protein, ORF 57-EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, ORF 57-EGFP was capable of shuttling from a transfected monkey nucleus to a recipient mouse nucleus in an interspecies heterokaryon assay. These data indicate that HHV-8 ORF 57 and IE63 possess certain common properties.

Human herpesvirus 8 primary infection occurs during childhood in Cameroon, Central Africa.

While in the United States and northern Europe, human herpesvirus 8 (HHV-8) appears to be mainly sexually transmitted with primary infection occurring in adulthood, the modes of transmission remain unknown in East and Central Africa, where Kaposi's sarcoma (KS) is a long-standing endemic disease, occurring not only in adults but also in children. The aim of our present study was to determine the prevalence of HHV-8 infection in children from Yaounde, Cameroon, Central Africa. Specific antibodies directed against both latent and lytic HHV-8 antigens were detected and titrated, with an immunofluorescence assay using the KS-1 cell line, in the plasma of 258 children and adolescents, of 32 mother and child pairs and of 189 pregnant women. Two different HHV-8 DNA-specific sequences were searched in the buffy coat by PCR assays. The overall HHV-8 seroprevalence was 27.5% among these children and adolescents. In newborns, seroprevalence reached 46%, reflecting passive transmission of maternal IgG. This was followed by a marked drop. Then, beginning around 4 years of age, a regular increase of HHV-8 antibodies took place, reaching 39% in the 12- to 14-year age group and 48% above 15 years, a rate similar (54.5%) to that observed in pregnant women. PCR detection of HHV-8 sequences was negative in seronegative children and positive in the buffy coat in 17% of HHV-8-seropositive children, reflecting a low viral load in the peripheral blood. Our results establish that in Central Africa HHV-8 infection takes place during childhood by casual routes, in contrast to the sexual transmission observed in adults in northern Europe and the United States. We hypothesize that the lymphadenopathic form of KS seen in African children is related to an early and massive infection by HHV-8 in susceptible individuals.

Sequence analyses of human herpesvirus-8 strains from both African human immunodeficiency virus-negative and -positive childhood endemic Kaposi's sarcoma show a close relationship with strains identified in febrile children and high variation in the K1 glycoprotein.

Human herpesvirus-8 (HHV-8) DNA sequences have been identified in all forms of Kaposi's sarcoma (KS), a cancer found primarily in adult AIDS patients. We have identified HHV-8 strains in a rare human immunodeficiency virus (HIV)-negative form of KS, which is endemic in children in parts of sub-Saharan Africa. This was shown in Zambia, where we also had identified HHV-8 sequences in blood from HIV-negative febrile children without KS. In order to investigate the relationship of these Zambian strains to each other and to those from other forms of KS, we compared them to strains we have characterized from European AIDS KS (Denmark) and all published sequences from all forms of KS. Four distinct genomic regions were examined by PCR and sequencing: ORF26, ORF75, gH and K1. The results showed a distinct grouping of strains from both sets of Zambian children in all genomic regions studied, but which was most pronounced in the K1 glycoprotein gene. This gene was highly variable, encoding up to 25% amino acid sequence variation. In contrast, the Zambian groups were closely related to each other, with only 2% variation. Similar results were found in comparisons to the K1 sequences from HIV-positive febrile infants or KS children. The data raise the possibility that in areas where rare childhood endemic KS occurs, geographical variation in HHV-8 may relate to differences in virulence or transmission.

Molecular and Serological Examination of the Relationship of Human Herpesvirus 8 to Multiple Myeloma: orf 26 Sequences in Bone Marrow Stroma Are Not Restricted to Myeloma Patients and Other Regions of the Genome Are Not Detected

Human herpesvirus 8 (HHV-8) genomic sequences were recently detected by polymerase chain reaction (PCR) and in situ hybridization in bone marrow stromal cells grown from multiple myeloma (MM) patients, but not in cells from control subjects (Rettig et al, Science 276:1851, 1997). We sought to confirm these observations in our own group of MM patients (n = 30). DNA was extracted from adherent stromal cells grown under varying conditions and assayed for HHV-8 sequence using PCR to amplify the orf 26 (KS330) sequence (Chang et al,Science 266:1865, 1997), as initially reported. Samples from human control subjects (n = 25) were concurrently extracted and analyzed. After 30 cycles of amplification, we did not detect any positive samples. In a more sensitive nested PCR, samples from 18 of 30 (60%) MM patients were positive, at about the limit of detection, but orf 26 sequence was also amplified from 11 of 25 (44%) human control samples. However, PCR amplification from other regions of the viral genome (orf 72 and orf 75) was uniformly negative for all MM and control samples, despite equivalent sensitivity. Additionally, all sera from MM patients were negative for HHV-8 IgG by immunofluorescence. Our data do not support a role of HHV-8 in the etiology of MM but may suggest the presence of a related (KS330-containing) virus in MM patients and in some control subjects.This is a US government work. There are no restrictions on its use.

Molecular and Serological Examination of the Relationship of Human Herpesvirus 8 to Multiple Myeloma: orf 26 Sequences in Bone Marrow Stroma Are Not Restricted to Myeloma Patients and Other Regions of the Genome Are Not Detected

Human herpesvirus 8 (HHV-8) genomic sequences were recently detected by polymerase chain reaction (PCR) and in situ hybridization in bone marrow stromal cells grown from multiple myeloma (MM) patients, but not in cells from control subjects (Rettig et al, Science 276:1851, 1997). We sought to confirm these observations in our own group of MM patients (n = 30). DNA was extracted from adherent stromal cells grown under varying conditions and assayed for HHV-8 sequence using PCR to amplify the orf 26 (KS330) sequence (Chang et al,Science 266:1865, 1997), as initially reported. Samples from human control subjects (n = 25) were concurrently extracted and analyzed. After 30 cycles of amplification, we did not detect any positive samples. In a more sensitive nested PCR, samples from 18 of 30 (60%) MM patients were positive, at about the limit of detection, but orf 26 sequence was also amplified from 11 of 25 (44%) human control samples. However, PCR amplification from other regions of the viral genome (orf 72 and orf 75) was uniformly negative for all MM and control samples, despite equivalent sensitivity. Additionally, all sera from MM patients were negative for HHV-8 IgG by immunofluorescence. Our data do not support a role of HHV-8 in the etiology of MM but may suggest the presence of a related (KS330-containing) virus in MM patients and in some control subjects.This is a US government work. There are no restrictions on its use.

Squamous cell carcinomas after allogeneic bone marrow transplantation for aplastic anemia: further evidence of a multistep process.

Secondary solid tumors are rare events occurring in patients who underwent allogeneic marrow transplantation for aplastic anemia and Fanconi's anemia. Human herpes virus 8 (HHV8), Epstein-Barr virus (EBV), and human papillomaviruses (HPV) sequences have been found in squamous cell carcinoma (SCC) occurring in organ transplant recipients. The tumor suppressor gene p53 has been strongly linked to the occurrence of SCC in the nonimmunocompromised population. In eight patients with SCC, we searched for HHV8, EBV, varicella zoster virus, adenovirus, and HPV sequences from DNA extracted from selected areas of SCC. We also looked for p53 expression in those specimens as well as the presence of anti-p53 antibodies in the serum of these patients at the onset of SCC. In one patient, we found the presence of both HHV8 and EBV sequences, and in another patient we found HPV16 sequences. All five tumors that could be studied disclosed evidence of p53 accumulation, but none of the eight patients had anti-p53 antibodies in the sera. SCC developing in marrow transplant recipients seems to occur via a multistep process. Genetic predisposition may be present, as in patients with Fanconi's anemia. Transplantation-related factors, such as irradiation and chronic graft-versus-host disease, also have a role. In this article, we add two more potent risk factors: p53 alteration(s) and in some cases the presence of oncogenic viruses.

Detection and subtyping of human herpesvirus-8 in renal transplant patients before and after remission of Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a complication of renal transplantation. If the human herpesvirus-8 (HHV-8) causes KS, the virus should be present in all KS lesions and be drastically reduced or cleared from involved tissue on remission of the KS. Fourteen renal transplant patients with cutaneous KS, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of KS, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. HHV-8 DNA was detected in all the cutaneous KS and all the visceral KS samples, as well as a number of KS-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-KS cases than that found in African endemic KS cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low grade viral replication that coincides with clinical remission of the KS lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant KS.

THE IMMUNOGENIC GLYCOPROTEIN GP35/37 OF HUMAN HERPESVIRUS 8 IS ENCODED BY OPEN READING FRAME K8.1

60 Background: Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi's sarcoma (KS) and body-cavity based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in the BCBL derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters {Renne 1996}. Methods: A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular weight is reduced to 30-kDa by digestion with protein-N-glycosidase F (PNGase F). By searching the HHV-8 sequence for open reading frames (ORF) with the potential to encode this glycoprotein, a new reading frame was identified adjacent to ORF K8. This ORF, termed K8.1, was predicted to encode glycoprotein of 229 amino acids translated from a spliced mRNA. Recombinant K8.1 was regularly recognized by KS-patient sera in Western blots, and immunoaffinity purified antibodies to recombinant K8.1 reacted with gp35-37. Results: The immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1. Recombinant K8.1 (rK8.1) expressed in E. coli is currently used for studies of HHV-8 epidemiology in children and adolescents, healty blood donors, and different groups at risk for KS. In this study antibodies against rK8.1 were detectable in 3.3% (4/118) of blood donors and 90.5% (19/21) of AIDS-KS patients. Antibodies against rK8.1 were not detectable in children below the age of 10 (0/400), whereas 3/100 adolescents aged 10 - 14 and 6/100 aged 15 - 19 were anti-rK8.1 positive. Conclusions: Although antibodies against only one protein are unlikely to reveal the complete epidemiology of HHV-8, the age distribution observed would be compatible with an agent which is transmitted in the context of sexual activity.