Detection of human herpesvirus 8 DNA in serum from blood donors with HHV-8 antibodies indicates possible bloodborne virus transmission

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma. There is a high seroprevalence of HHV-8 in several African countries, but the transmission route is not known definitively. In this study 174 serum samples from blood donors in Tanzania were examined by immunofluorescence assays detecting antibodies to latent and lytic HHV-8 antigens. Real-time polymerase chain reaction was used for detection and quantification of HHV-8 DNA in serum. In all, 83/174 (48%) of the subjects had antibodies to latent or lytic antigens. Forty (23%) had antibodies to both antigens and of those eight (20%) had detectable HHV-8 DNA in serum. HHV-8 DNA load correlated with antibody titres to lytic, but not latent, HHV-8 antigens. This supports the usefulness of anti-lytic antibodies in HHV-8 serology and suggests that transmission of HHV-8 by blood contact could be of importance in this region.     

Seroprevalence of human herpes virus 8 in different Eritrean population groups.

BACKGROUND: Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma. In the US and Europe, HHV-8 is believed to be mainly sexually transmitted, but reports from some African countries suggest non-sexual transmission. OBJECTIVES: To find out more about HHV-8 seroprevalence and transmission in Eastern Africa. STUDY DESIGN: In this study, 411 serum samples from different population groups in Eritrea (children, pregnant women, female sex workers and members of the isolated Rashaida tribe) were examined for HHV-8 antibodies with an immunofluorescence assay detecting antibodies to latent and lytic HHV-8 antigens. RESULTS: Antibodies to HHV-8 latent antigen were found in 0-2% of Eritrean children, 5% of pregnant women, 8% of female sex workers and 26% of Rashaidas, respectively. No correlation was found between detectable HHV-8 antibodies and seropositivity to HIV or herpes simplex 2. CONCLUSIONS: These results suggest that HHV-8 infection is relatively common in Eritrea and that viral transmission occurs predominantly through non-sexual route in this region.     

Human herpesvirus 8 seroprevalence and evaluation of nonsexual transmission routes by detection of DNA in clinical specimens from human immunodeficiency virus-seronegative patients from central and southern Italy, with and without Kaposi's sarcoma

In order to investigate the seroprevalence of human herpesvirus 8 (HHV-8) infection in central and southern Italy, sera from human immunodeficiency virus (HIV)-seronegative subjects, with and without Kaposi's sarcoma (KS), were analyzed by immunofluorescence assay, using BC-3, a cell line latently infected with HHV-8. High titers of antibody against HHV-8 lytic and latent antigens were detected in all 50 KS patients studied, while in 50 HIV-seronegative subjects without KS, 32 (64%) were found positive for HHV-8 antibodies. Titers in the sera of these patients were lower than those for KS patients. This data suggests that HHV-8 infection is not restricted to KS patients and that the prevalence of HHV-8 infection in the general population may be correlated with differing rates of prevalence of KS in different parts of the world. In view of these findings, possible nonsexual transmission routes were evaluated. Nested PCR was used to test for the presence of HHV-8 DNA in saliva, urine, and tonsillar swabs from KS and non-KS patients. In KS patients, 14 out of 32 tonsillar swabs (43.7%), 11 out of 24 saliva samples (45.8%), and just 2 out of 24 urine samples (8.3%) tested positive for HHV-8 DNA. In the control group, on the contrary, none of the 20 saliva and 20 urine specimens was positive for HHV-8 DNA; only 1 out of 22 tonsillar swabs gave a positive result. This data supports the hypothesis that HHV-8 infects the general population in a latent form. The reactivation of viral infection may result in salivary shedding of HHV-8, contributing to viral spread by nonsexual transmission routes.     

Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) infection in men receiving treatment for HIV-1 infection

OBJECTIVE: To determine the prevalence of human herpesvirus 8 (HHV-8) infection in men treated for HIV-1 infection in Denver, Colorado. DESIGN: Cross-sectional analysis METHODS: Blood samples were obtained from 216 HIV-1-infected men. Antibody to latency-associated nuclear antigen (LANA) was detected by an immunofluorescent assay and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC) was detected by polymerase chain reaction amplification. RESULTS: Among HIV-1-infected men who did not have Kaposi's sarcoma (KS), prevalence of HHV-8 infection was 46% (95% confidence interval [CI], 0.39-0.52). LANA seropositivity was common both among subjects with KS and subjects without KS (69% versus 42%; p = .06), but detection of HHV-8 DNA in peripheral blood was strongly associated with a diagnosis of KS (44% versus 10%; p = .001). In a univariate analysis of study subjects without KS, neither the odds of LANA seropositivity nor detection of HHV-8 DNA in PBMC was significant for CD4+ lymphocyte count, HIV-1 virus load, the use of three drug antiretroviral regimens or the prior occurrence of non-KS AIDS-related conditions. CONCLUSION: Although antibodies to HHV-8 are common among HIV-1-infected men, detection of HHV-8 DNA in PBMC is uncommon and is associated with a diagnosis of Kaposi's sarcoma.     

Antibodies to human herpesvirus 8 latent and lytic antigens in blood donors and potential high-risk groups in Sweden: variable frequencies found in a multicenter serological study

Human herpesvirus 8 (HHV-8) is a herpesvirus associated with Kaposi's sarcoma (KS). An immunofluorescence assay was used for detection of IgG, IgM, and IgA antibodies against lytic and latent HHV-8 antigens to analyse samples from KS patients (n = 8), healthy blood donors (n = 162), individuals with a high risk sexual behaviour (n = 114), and bone marrow transplant patients (with high risk for bloodborne infections) (n = 34) in Sweden. Of the KS patients, 88% had IgG antibodies to both lytic and latent antigens by immunofluorescence. In all other groups, antilatent antibodies were rare (0-2.6%). IgG antibodies to the lytic antigens were found, by immunofluorescence, in 20% of the blood donors, 31% of the high risk patients, and in 24 and 29% of the bone marrow transplant patients (pre- and post-transplant samples, respectively). For verification of the specificity of the anti-lytic antibodies, 170 of the samples were also tested blindly at different laboratories world-wide with five other assays shown previously to detect HHV-8 antibodies in most KS patients. By using two recombinant HHV-8 proteins (ORF65/vp17 and K8.1/gp 35-37) in ELISA, a whole-virion ELISA and two immunofluorescence assays confirmation of the reactivity against lytic viral antigens was sought. The comparison of the different methods suggested the K8.1 ELISA to be highly specific and also showed a good agreement between two of the immunofluorescence assays. However, generally there was a poor correlation for positive results, indicating the need of further methodological development.     

Detection of human herpesvirus 8 DNA and antibodies to latent nuclear and lytic-phase antigens in serial samples from aids patients with Kaposi's sarcoma.

BACKGROUND: human herpesvirus 8 (HHV-8) have recently implicated in the etiology of Kaposi's sarcoma (KS), but the pathophysiologic and immunologic interactions between HHV-8 and the human host are incompletely understood. OBJECTIVE: this paper intends to present partial results of a follow-up study of KS patients, designed to investigate HHV-8 viremia and antibody response. METHODS: ninety-six paired serial samples (PBMCs and sera) were obtained from 12 aids patients with KS who received HAART prior or just after entry in the study. HHV-8 DNA was detected by nested-PCR and antibodies to HHV-8 latent nuclear antigen (LANA) and lytic antigen by immunofluorescence assay (IFA). RESULTS: HHV-8 DNA was detected in 33.3% of the first PBMC samples. Among the eight PCR negative patients, four presented positive samples during the follow-up and four remained negative. Five patients had intermittent viremia. Fifteen of the 96 PBMC samples were PCR positive (15.6%). Four of 39 samples (10.2%) from patients classified as stadio II and 11 of the 53 samples (20.7%) from patients in stadio IV were PCR positive (P=0.2). Six patients (50%) had anti-LANA antibodies at the entry in the study. Among the six seronegative patients, two seroconverted 2 months later and four patients remained seronegative during the 5-8 months of follow-up. All patients had anti-lytic antibodies since the first sample. CONCLUSION: the presence of HHV-8 viremia could be related to the severity of KS and could be intermittent even under HAART. A longer follow-up is needed to confirm these results.     

Human herpesvirus 8 seroconversion in Kenyan women by enzyme-linked immunosorbent assay and immunofluorescence assay.

BACKGROUND: Human herpesvirus 8 (HHV-8) antibody tests vary in reported sensitivity and specificity, depending on the population tested and the assay. OBJECTIVE: The purpose of this study was to compare the ability to detect seroconversion to HHV-8 in a cohort of HHV-8 seronegative female commercial sex workers in Kenya using three tests: HHV-8 viral lysate-based enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay for HHV-8 lytic antigens (IFA-lytic) and IFA for latent nuclear antigens (IFA-LANA). STUDY DESIGN: By ELISA, 16 women from a prospective cohort of commercial sex workers were identified as seroconverting to HHV-8. A total of 124 post-enrollment samples from these 16 women as well as the enrollment samples were tested for HHV-8 antibodies by all three assays to monitor seroconversion. RESULTS: Of 16 women with apparent seroconversion by ELISA, 8 had a rise in IFA-lytic titers either concomitant with or prior to the first positive ELISA sample and no initial LANA by IFA. Five of the 16 women were IFA-LANA positive at entry, indicating prior infection with HHV-8. Three women had no evidence of seroconversion by either IFA-lytic or IFA-LANA and two of these three had increased ELISA reactivity concomitant with HIV-1 infection. CONCLUSIONS: Conversion from a negative to a positive ELISA result for HHV-8 antibody indicated seroconversion in only half of the study cohort of 16 women when IFA-lytic and IFA-LANA results were considered. The IFA-lytic assay was more sensitive than ELISA for early antibody responses. The IFA-LANA was positive in some women who had neither IFA-lytic nor ELISA antibodies suggesting it may be a marker for latent infections. Presumptive identification of incident HHV-8 infection by ELISA screening followed by IFA-lytic testing to confirm the positive test and IFA-LANA to rule out prior infection provides the most accurate documentation of HHV-8 seroconversion.     

High seroprevalence of human herpesvirus 8 (HHV-8) in HIV-1-infected pregnant women of Southeastern Italy: association with injection drug use and hepatitis C virus infection

The seroprevalence of human herpesvirus 8 (HHV-8) in a group of HIV-1-infected pregnant women and in mother-child pairs from Southeastern Italy (Apulia) was determined. Blood was collected from 49 HIV-1-infected women during pregnancy or at delivery as well as from their children. Samples were analysed for the presence of antibodies to the latency-associated nuclear antigen and a structural antigen encoded by open reading frame 65. The presence of antibodies to hepatitis C virus (HCV) was also determined. Nineteen women (38.7%) were found to be positive for HHV-8 antibodies to at least one of the two antigens, and 21 (42.9%) for HCV antibodies. HHV-8 antibodies were more common in injecting drug users (56.3%) than in women infected through heterosexual intercourse (30.3%). HCV antibodies were significantly more prevalent in HHV-8-seropositive (66.7%) than HHV- 8-seronegative (29%) women. Thirteen children born to HIV-1/HHV-8 co-infected women were HHV-8-seroreactive, with a variable pattern of reactivity to the analysed antigens. Follow-up of children showed a prolonged persistence of antibodies, in two cases for more than 12 months. This study has provided serological evidence for a high rate of HHV-8 infection in HIV-1-infected women in the Apulia region, and has identified a possible association between HHV-8 infection, past use of injection drugs and HCV infection. Parenteral transmission may, therefore, be a mode of virus spread.     

No increased human herpesvirus 8 seroprevalence in patients with HIV-associated non-Hodgkin's lymphoma

Human herpesvirus 8 (HHV-8) is closely associated with Kaposi's sarcoma (KS), HIV-associated Castleman's disease, and primary effusion lymphoma. As a high frequency of non-Hodgkin's lymphoma (NHL) has been reported in patients with HIV-associated KS, we hypothesized that HHV-8 infection could be indirectly implicated in the pathogenesis of NHL. We assessed the prevalence of HHV-8 antibodies in 63 patients with NHL compared with 126 HIV-infected matched control patients without NHL. Serum samples from cases and controls were assayed for antibodies to HHV-8 lytic and latent antigens using an indirect immunofluorescence assay. In patients with concordant serologic results, HHV-8 antibodies were detected in 41.5% of the NHL cases and 37% of the controls. This absence of a significant difference in HHV-8 seroprevalence between cases and controls (p =.73) does not support a possible role for HHV-8 infection in the development of NHL in HIV-infected patients.     

Seroprevalence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 in several regions of Italy

OBJECTIVE: To study the seroprevalence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8) in 779 Italian blood donors. STUDY DESIGN/METHODS: Sera were tested for antibodies to a latency-associated nuclear antigen (LANA) and a capsid related protein encoded by ORF65. RESULTS: Among all Italian donors, 17.7% and 18.7% had antibodies to LANA and ORF65 protein, respectively, and 24.1% had antibodies to at least one antigen. KSHV/HHV-8 seroprevalence was higher in the Po valley and in Sardinia than close to the sub-Alpine Veneto region, Tuscany, or Apulia. KSHV/HHV-8 seroprevalence was almost equally distributed between men and women but increased in the older age groups. CONCLUSIONS: The regional differences and age distribution in seroprevalence agree partially with the incidence of classic KS in Italy. The rarity of classic KS in KSHV/HHV-8-infected subjects and the equal gender distribution of seroprevalence suggest that other cofactors may contribute to KS development in human immunodeficiency virus type 1 (HIV-1)-uninfected individuals.     

Colocalization of the viral interleukin-6 with latent nuclear antigen-1 of human herpesvirus-8 in endothelial spindle cells of Kaposi's sarcoma and lymphoid cells of multicentric Castleman's disease

Human herpesvirus-8 (HHV-8) also called Kaposi's sarcoma-associated herpesvirus infects spindle cells in Kaposi's sarcoma (KS) and lymphoid cells in multicentric Castleman's disease (MCD). In KS cells, HHV-8 is mainly latent with the expression of latent nuclear antigen-1 (LNA-1), whereas in MCD both lytic and latent antigens are produced by lymphoid cells. We show by immunohistochemical labeling that in KS viral interleukin-6 (vIL-6) is expressed in rare spindle cells, whereas in MCD, vIL-6 is detectable in lymphoid cells around lymphoid follicles but also within the follicular dendritic reticulum cell network. The staining of apoptotic bodies with anti IL-6 antibody suggests the achievement of a complete lytic cycle in a subset of lymphoid cells. Interestingly, in MCD, some areas contained vascular spindle cells latently infected by HHV-8 on the basis of LNA-1 expression. This finding might imply that in MCD, both vascular and lymphoid cells proliferate in response to the viral infection. Double immunostaining with anti LNA-1 and anti vIL-6 in MCD and KS identifies 2 subsets of HHV-8 infected (vascular and lymphoid) cells, some with exclusive expression of LNA-1 and some with coexpression of vIL-6 and LNA-1. This suggests that in vivo the regulation of the expression vIL-6 and LNA-1 protein varies with the cell type. In addition, the detection of infected endothelial cells in MCD may indicate that these cells belong to the reservoir for HHV-8.     

Kaposi's sarcoma-associated herpesvirus seroprevalence in selected German patients: evaluation by different test systems

A total of 603 serum samples obtained from 12 different patient and control groups, including potentially cross-reactive sera, were tested for the presence of antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8). The assays used were an inhouse immunofluorescence test (IFT) employing latent KSHV antigens and a prototype enzyme-linked immunosorbent assay (ELISA) coated with recombinant latency-associated KSHV nuclear antigen (LANA, open reading frame 73) and the K8.1 protein. Sera giving discrepant results were additionally tested with two commercial IFTs employing KSHV latent and lytic antigens, respectively. The low KSHV seroprevalence rate found in blood donors (3.0% by in-house IFT, 2.0% by recombinant ELISA) was comparable to that found previously in Western European countries. The highest KSHV seroprevalence rates were found in patients with Kaposi's sarcoma (100% by both assays), followed by HIV-infected men without Kaposi's sarcoma (23.3% by in-house IFT and 17.8% by ELISA) and women (15.7% by in-house IFT and 13.7% by ELISA). Overall correlation between both assays was 91.2%, with the highest rate of discordant results occurring in HIV-infected male subjects. Retesting of the 53 discrepant samples by the commercial IFTs revealed the best, albeit low, correlation between the in-house IFT and the commercial latent antigen IFT and poor correlations between the other assays. Apart from patients with autoimmune antibodies, there was no significant degree of non-specific reactivity in either of the KSHV tests due to antibodies against Epstein-Barr virus and human herpesvirus 6. Despite the lack of a "gold standard" for KSHV antibody detection, the fact that the results obtained overall agreed rather well indicates their suitability for conducting seroepidemiological studies.     

Distinct patterns of viral antigen expression in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus coinfected body-cavity-based lymphoma cell lines: potential switches in latent gene expression due to coinfection.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens approximately 110 and 41-42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.     

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.     

Evaluation of BC-3 and BCP-1 cell lines in immunofluorescence assay to detect HHV-8 antibodies in Kaposi's sarcoma, multiple myeloma and other groups.

BACKGROUND: We describe a comparative study of an immunofluorescence assay using inducible BC-3 and BCP-1 cell lines as sources of HHV-8 antigens. STUDY DESIGN: Detection of both antibodies to proteins expressed in lytic cycle and during latency in sera from HIV-infected patients with KS, HIV-positive patients without KS, normal blood donors, HIV-negative pregnant women and HIV-negative patients with multiple myeloma. Where possible, detection of antibody was associated with nested PCR detection of HHV-8 in peripheral mononuclear cell (PBMC) samples collected from AIDS-KS patients. RESULTS: Immunofluorescence was more intense with the BC-3 cell line than with BCP-1, thus facilitating examination under the microscope. HHV-8 antibodies were detected among 82.75% of AIDS-KS patients, in 27.3% of HIV-infected homosexual men, 2% of blood donors and in 2% of pregnant women. No HHV-8 antibodies were detected in serum samples from HIV-negative patients presenting multiple myeloma. HHV-8 DNA sequences were detected and confirmed by southern blot hybridization in five out of 17 (29.4%) PBMC samples from AIDS-KS patients. Titre of antibodies to proteins expressed in lytic cycle was much higher than the titre of antibodies to proteins expressed during latency. CONCLUSIONS: Both immunofluorescence assays were found useful and HHV-8 seroprevalence rates reported in previous studies were confirmed. In addition, results obtained using these assays tend to provide evidence for a lack of epidemiological association between HHV-8 infection and development of multiple myeloma.     

High diversity of HHV-8 molecular subtypes in the Amazon region of Brazil: evidence of an ancient human infection

The present study describes the molecular epidemiology of Human herpesvirus 8 (HHV-8) among four Indian tribes (Kararao, Arara Laranjal, Tiriyo, and Zo'e) of the Amazon region of Brazil and a group of HIV-1-infected subjects from the urban population of Belem, Para. Infection was characterized by the presence of antibodies using ELISA (measuring antibodies to ORF59, ORF65, K8.1A, K8.1B, and ORF73), and molecular assays (gene amplification of the regions ORF26 and the variable region VR1). Antibodies to HHV-8 were detected in 66 samples of the 221 Brazilian Amerindians, namely, 6 (25%) in the Kararao, 18 (19.6%) in the Arara Laranjal, 24 (42.9%) in the Tiriyo, and 18 (36.7%) in the Zo'e. Among the 477 HIV-1-infected subjects, antibodies to HHV-8 were present in 74 (15.5%) persons. The ORF26 region was amplified in seven samples, one of the Arara Laranjal, one of the Tiriyo, two of the Zo'e, and three of the HIV-1-infected group. Subtyping of HHV-8 described a high multiplicity of molecular subtypes, including C (Zo'e), E (Tiriyo), and B (HIV-1 infected). Serological results confirm the high prevalence of HHV-8 among Amerindians and the presence of three subtypes in the Amazon region of Brazil, including a unique subtype, which favors the idea of HHV-8 as an ancient human infection within this particular geographical region.     

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi’s sarcoma

 Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi’s sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8. Received: 3 August 1996 / Accepted: 28 November 1996     

Distinct expression of Kaposi's sarcoma-associated herpesvirus-encoded proteins in Kaposi's sarcoma and multicentric Castleman's disease

The expression of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)-encoded proteins is herein demonstrated in Kaposi's sarcoma (KS) and multicentric Castleman's disease (MCD) in a single lymph node derived from a patient with acquired immunodeficiency syndrome. Immunohistochemistry revealed that both lytic and latent KSHV proteins were expressed in cells of the MCD lesion. KSHV-encoded viral interleukin-6 was also detected in follicular dendritic cells of the germinal center. Cytoplasmic localization of open reading frame 59 protein and latency-associated nuclear antigen suggested KSHV activation in the MCD lesion. Moreover, a high copy number of KSHV was detected in the blood. Clinically, pegylated-liposomal doxorubicin induced regression of not only KS, but also lymphadenopathy of the MCD lesion with a decrease in KSHV load and human interleukin-6 in the blood. To the best of the authors' knowledge this is the first case demonstrating differential expression of virus proteins in two KSHV-associated diseases, KS and MCD, in the same section. The case confirms lytic KSHV infection in MCD, and suggests that clinical symptoms of MCD might be closely linked with KSHV activation.     

Comparative evaluation of three serological methods for detection of human herpesvirus 8-specific antibodies in Canadian allogeneic stem cell transplant recipients

Human herpesvirus 8 (HHV-8) has been associated with all types of Kaposi's sarcoma (KS), including posttransplantation KS. However, little is known regarding HHV-8 infections in hematopoietic stem cell transplant (SCT) recipients. In this study, we used a variety of serological assays, including in-house-developed enzyme immunoassays (EIAs) utilizing synthetic peptides corresponding to lytic viral antigens (ORFs 65 and K8.1) and a commercial EIA kit based on a whole virus lysate (Advanced Biotechnologies Inc.), as well as latent- and lytyc-antigen-based immunofluorescence assays (IFAs) to determine the seroprevalence of HHV-8 in 42 allogeneic SCT recipients from Canada. Using the two peptide-based EIA methods as for screening, HHV-8-specific antibodies were detected in five (12%) patients between days 21 and 91, although only one (2%) subject was positive for HHV-8-specific antibodies before transplantation. All positive results from these five patients were confirmed by at least one of the IFAs, with an additional patient showing seropositivity before transplantation. However, the commercial EIA was negative at all time points (days -7, 21, and 91) in those five patients. The episodes of seroconversion or reactivation were not associated with sustained viremia, since HHV-8 DNA was not detected by real-time PCR in the corresponding leukocytes and plasma of the seropositive patients. No clinical or laboratory abnormalities were clearly associated with HHV-8 seropositivity. This study confirms the utility of simple peptide-based EIA methods to assess the presence of HHV-8-specific antibodies in immunocompromised patients and emphasizes the need of conducting prospective studies to determine the source of HHV-8 infection in SCT recipients.