Age-specific seroprevalence of Human Herpesvirus 8 in Mediterranean regions

Objective   Human herpesvirus 8 (HHV8) is believed to be transmitted mainly by sexual contact; epidemiological data from Africa show, however, that non-sexual transmission routes may also play an important role. To evaluate better the distribution of HHV8 infection in the Mediterranean area, we performed an age-specific seroprevalence study.
Methods   Sera were collected from subjects from different geographical areas. The sera were analyzed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). A total of 1083 patients were studied, 667 patients from various regions of Italy and 416 from Albania. The patients were stratified into six age groups. Multivariate logistic regression was used to evaluate associations between HHV8 and demographic data.
Results   An overall seropositivity rate of 17.6% was observed. The highest rate was observed in Sardinia (25.0%) and the lowest was found in Albania (13.9%). The prevalence rate increased linearly with age, from 9.7% in patients belonging to the 0–14 years age group to 26.3% for patients more than 59 years old. Seropositivity for HHV8 was significantly associated with membership of the 59 years-plus age group. Rates of seropositivity were significantly higher in patients from central southern Italy (OR = 1.7) and Sardinia (OR = 1.8) than in patients from Albania.
Conclusions   The data suggest that HHV8 is widespread in the Mediterranean area, including regions like Albania that have not been previously investigated. The statistically significant association between HHV8 seropositivitity and increasing age suggests that non-sexual transmission routes may be involved in the spread of the virus.     

Laboratory Diagnosis of Human Herpesvirus 8 Infection in Humans

Human herpesvirus 8 (HHV-8) is causally associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Serological and molecular biology assays are used to investigate the biology of this virus in different populations and diseases. Serological assays are mainly used to study the prevalence of the viral infection and to predict the diagnosis of Kaposi's sarcoma and other HHV-8-associated cancers. The appearance of antibodies against lytic antigens precedes the appearance of antibodies against latent antigens, probably explaining the lower sensitivity of assays based on latent HHV-8 antigens. The lack of international reference serum panels is presently the major bottleneck for further progress in the field of HHV-8 serology. Molecular biological assays are an absolute requirement for both the diagnosis and the follow-up of HHV-8 infection. Qualitative methods have been particularly useful to elucidate the mode of transmission and the causal association between HHV-8 and HHV-8-associated diseases. Quantitative methods have become an essential tool to monitor the progression of the infection and the effects of antiviral therapies. This review analyzes the performance of the different serological and molecular biological assays available at present. The main conclusion is that more research is needed to define the most useful laboratory tests for the diagnosis of HHV-8 infection and to establish the clinical role of such tests. Electronic Publication     

Human Herpesvirus 8 and Protection from AIDS Dementia Complex

Infection with human herpesvirus 8 (HHV-8) has been associated with the development of three distinct conditions: Kaposi's sarcoma, body cavitybased lymphoma and Castleman's disease. HHV-8 produces chemokinelike proteins including viral macrophage inflammatory protein II, which has been shown to block human immunodeficiency virus 1 (HIV-1) infection of CD4-positive cells expressing CCR-3. As CCR-3 is a receptor for HIV-1 into microglial cells, it has been hypothesized that HHV-8 infection may inhibit HIV-1 infection of the brain, thereby decreasing the incidence of AIDS dementia complex. We reviewed published studies of the incidence of AIDS dementia complex in individuals with and without Kaposi's sarcoma. The data are consistent in showing a negative association between Kaposi's sarcoma and AIDS dementia complex and, although sparse, support the hypothesis that productive HHV-8 infection decreases HIV-1 infection of the brain sufficiently to decrease the incidence of AIDS dementia complex. This negative association should be examined in further cohorts of HIV-1-infected subjects, to exclude alternative explanations.     

Lytic Growth of Human Herpesvirus 8: Morphological Aspects

The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a gamma herpesvirus associated with AIDS-related body cavity-based lymphomas (BCBL), also called primary effusion lymphomas (PEL). These are a rare form of non-Hodgkin lymphomas in which HHV-8 is present, often associated with Epstein-Barr virus (EBV) infection. HHV-8 is also present in a latent state or in a state of low-level persistence in different primary effusion lymphoma-derived cell lines, such BCBL-1 cells, that lack EBV infection. This cell line was induced to produce mature virions by treatment with 12- O -tetradecanoyl phorbol-13-acetate (TPA) and the characteristic ultrastructural features of HHV-8 lytic replication were identified and compared to those of the other members of Herpesviridae family.     

New Immunofluorescence Assays for Detection of Human Herpesvirus 8-Specific Antibodies

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a “gold standard.” Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (κ > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV⁺) Kaposi's sarcoma (KS) patients, HIV⁺ KS⁻ patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.     

Seroprevalence of rubella among women of childbearing age in Switzerland

A seroepidemiological study was carried out in Switzerland to define the population susceptible to rubella among women of childbearing age. IgG antibodies to rubella virus were determined in 9,046 women giving birth between 1 August 1990 and 30 September 1991 in 23 of 26 Swiss cantons. These sera represented 10–20 % of the yearly total number of births in each Swiss canton. Anti-rubella IgG was measured by an automated enzyme-linked fluorescent assay for use with a commercial system (Vidas Rub IgG, bio-Mérieux, France). Before the study population was screened, the commercial system was compared to the traditional hemagglutination-inhibition (HAI) test using 500 consecutive samples from parturient women. The sensitivity was 97.7 %, the specificity was 100 %, and agreement between the two tests was 97.8 %. The discrepancies corresponded to very low titres of antibodies as measured by HAL The seroprevalence of rubella nationwide in women of childbearing age in Switzerland was 94.3 %. The seroprevalence was higher (96.5 %) in the 5,677 women of Swiss nationality than in the 3,090 women of a different nationality (90.4%) (p<0.001). In Swiss women the seroprevalence of rubella did not increase significantly with age and was identical in primiparous and in multiparous women, thus indicating that women of childbearing age are probably not sufficiently immunised.     

Comparison of Serologic Assays for Detection of Antibodies against Human Herpesvirus 8

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.     

Four-Antigen Mixture Containing V-Cyclin for Serological Screening of Human Herpesvirus 8 Infection

Improved diagnostic reagents and testing are currently needed for the serological detection of human herpesvirus 8 (HHV-8) infections. We evaluated the luciferase immunoprecipitation systems (LIPS) for profiling antibody responses to a panel of HHV-8 proteins for diagnosis of Kaposi sarcoma (KS)-infected individuals. Using a pilot serum set, LIPS detected robust antibody responses to several known antigens, and a screen of 14 additional HHV-8 proteins identified v-cyclin as a potentially new diagnostic antigen. In evaluating a training-serum set, a four-antigen panel (K8.1, v-cyclin, ORF65, and a LANA fragment) was found to provide sufficient information for diagnosis. Analysis of a validation serum set using the combined results from these four separate antigen tests showed 100% sensitivity and 100% specificity. Furthermore, a LIPS format using a mixture of the four antigens, which simplifies data collection and analysis, closely matched the diagnostic performance of the combined separate tests (R = 0.95). This four-antigen mixture format analyzed with the validation serum set also showed 100% sensitivity and 100% specificity but was not statistically different from two separate enzyme-linked immunosorbent assays (94% sensitivity and 100% specificity) using baculovirus-produced LANA and bacterially produced K8.1. Heat map analysis of KS patient antibody titers revealed marked heterogeneity in humoral responses to this four-antigen panel. Overall, the LIPS assay showed 97% sensitivity, and positive anti-v-cyclin antibodies were detected in approximately 75% of the KS sera. These results suggest that LIPS screening using an antigen mixture is a sensitive and high-throughput method for serological screening of HHV-8 infection in individuals with KS.Kaposi sarcoma (KS) is an opportunistic disease in human immunodeficiency virus (HIV) patients and the most common cancer associated with AIDS worldwide (12). Identified a decade ago as the causative agent of KS, human herpesvirus 8 (HHV-8), also known as Kaposi''s sarcoma-associated herpesvirus, has an approximately 165-kb genome encoding about 90 gene products (21). Many of these gene products allow the virus to evade the human immune system (8). KS primarily affects AIDS patients, but it can also occur in non-HIV-infected individuals and presents as classical, endemic, and posttransplant forms. HHV-8 is also associated with two other rare B-cell lymphoproliferative disorders, primary effusion lymphoma and multicentric Castleman disease, which are primarily found in HIV-infected or other immunosupressed patients.Currently, there is a need for sensitive and specific testing to identify HHV-8-infected individuals, especially among potential blood donors (14). Low viral loads in blood limit the sensitivity and thus the usefulness of PCR-based approaches (20). Alternatively, a variety of serological tests, including immunofluorescence assays (23), Western blotting (26), and enzyme-linked immunosorbent assays (ELISAs) (11, 15, 18), have been employed to detect antibodies to HHV-8 proteins and to diagnose infection. Considerable progress has been made in employing defined recombinant HHV-8 antigens, including LANA, K8.1, ORF65, for testing. The most sensitive ELISAs require separate determinations of two or three HHV-8 antigens and typically rely on diagnostic algorithms to achieve 90 to 95% sensitivity and 90 to 95% specificity at best (15, 18). Furthermore, one major problem plaguing the assessment of the performance of any given HHV-8 serological test is the lack of gold standard reference serum samples (19). Typically KS patients are the only true positives available, which may cause the sensitivity of the assay to be overestimated, because KS patients generally have much higher antibody titers than asymptomatic HHV-8-infected individuals (13).Recently, Renilla luciferase (Ruc)-antigen fusions, produced in Cos1 cells, were used in a simple immunoprecipitation assay called the luciferase immunoprecipitation systems (LIPS) to quantitatively measure antibody responses to cancer-associated autoantigens (2), autoantigens associated with autoimmune diseases (3, 4), and a variety of infectious agents, including hepatitis C virus (1), HIV (1), human T-cell leukemia virus type I (6), herpes simplex virus types 1 and 2 (5), and filarial infections (7, 24). LIPS is based on fusing protein antigens to a light-emitting enzyme reporter, Ruc, and expressing these fusions in mammalian cells. The Ruc-labeled antigen extracts are then used in immunoprecipitation assays with serum samples and protein A/G beads. Following washing, light production is measured, yielding highly quantitative antibody titers. In this study, we used LIPS to evaluate known antigens and potential HHV-8 ORFs for the serological diagnosis of KS. Following the evaluation of pilot and training-serum sets, a four-antigen panel (K8.1, v-cyclin, ORF65, and LANA fragment) was selected. This four-antigen panel, evaluated separately or as a mixture with a validation serum set, showed 100% sensitivity and 100% specificity. These results suggest that a LIPS antigen mixture is an efficient high-throughput method for serological screening of HHV-8 infection in individuals with KS.     

Viral Load of Human Herpesvirus 8 in Peripheral Blood of Human Immunodeficiency Virus-Infected Patients with Kaposi's Sarcoma

Viral load is an important marker of activity of viral diseases for a number of viruses. We wished to evaluate whether the viral load of human herpesvirus 8 (HHV-8) in peripheral blood was a consistent feature of Kaposi's sarcoma (KS) patients and whether the viral load correlated with human immunodeficiency virus (HIV) RNA levels, CD4 counts, and/or the HHV-8 seroreactivity. Fifty-four consecutive plasma samples from 14 patients with KS were evaluated for HHV-8 viral load by quantitative real-time PCR. Samples were analyzed at the start of highly active antiretroviral therapy (HAART) and at different intervals during treatments. The median HHV-8 DNA load before HAART treatment was 8,998 (ranging from 170 to 40,100) copies/ml and 12,270 (ranging from 40 to 142,575) copies/ml during HAART. There were both increasing and decreasing trends. There was an association between HHV-8 DNA and HIV RNA viral loads (odds ratio [OR] = 5.40; 95% confidence interval [95% CI], 1.54 to 18.98) and between HHV-8 viral load and CD4 cell counts (OR = 7.24; 95% CI, 1.30 to 40.35). High HHV-8 viral load was also correlated with the titers of antibodies to the lytic HHV-8 antigen detected with immunofluorescence (P < 0.01), but not with antibodies to the latent HHV-8 antigen. In conclusion, we found that HHV-8 viremia in KS is associated with HIV viral load, CD4 cell counts, and lytic HHV-8 serological reactivity. HHV-8 viral load monitored by real time PCR might be useful for determination HHV-8 viral load during the follow-up of KS patients.     

Kaposi's Sarcoma and Human Herpesvirus 8 in Cuba: Evidence of subtype B expansion

Objective

To evaluate the temporal distribution (1991-2009) and associated variation of KSHV subtypes in Cuba.

Method

Phylogenetic characterization based on the KSHV K1 gene was performed using 90 KSHV positive samples.

Results

Molecular characterization confirmed the prevalence of a wide range of KSHV subtypes (A: n=48 [A5=12]; C: n=15; B: n=22; and E: n=5). In the current study, we observed a significant increase in HHV-8 subtype B after 2004 (p=0.0063). This Subtype B in Cuba was associated with: heterosexual behaviour (OR: 3.63, CI: 1,2-10,98; p=0.03), with the antecedent of acquiring HIV/KSHV in Africa (p=0.0003), with nodular stage of KS lesions (OR 4.2, CI: 1.1 to 15.7; p=0.04).

Conclusion

Our study is the first to report KSHV Subtype B expansion in Cuba, that might be reflective of a change in human behavioural pattern.     

应用捕获再捕获法估计2006年深圳市男同性恋人群规模

目的调查估计深圳市男同性恋人群规模，分析影响评估结果的因素，为制定有针对性的艾滋病防治策略提供科学依据。方法应用捕获-再捕获法对深圳市6个男同性恋活动场所进行调查估计。结果深圳市约有男同性恋57261-89235人。结论运用捕获-再捕获法进行估计，用时短，花费较低，但受到捕获周期、场所聚集性的影响，调查研究时应充分考虑。     

Monofunctional and Polyfunctional CD8+ T Cell Responses to Human Herpesvirus 8 Lytic and Latency Proteins

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease. It is postulated that CD8⁺ T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8⁺ T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8⁺ T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8⁺ T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.Human herpesvirus 8 (HHV-8), also referred to as Kaposi''s sarcoma-associated herpesvirus, is a gammaherpesvirus that causes Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease. The importance of developing effective prevention and treatment for HHV-8 infection is evident in that KS, a neoplasm of endothelial origin, continues to be the most common cancer among human immunodeficiency virus (HIV)-infected patients (8). KS is also the leading cause of cancer in children in sub-Saharan Africa (7). Although the incidence of KS in HIV-infected persons declined with the advent of antiretroviral therapy (ART) (10), KS can occur in persons on ART with suppressed HIV infection and high CD4⁺ T cell counts (25).The immune responses responsible for controlling HHV-8 infection and preventing KS are not clear. CD8⁺ T cell immunity likely plays a significant role in HHV-8 infection, as these cells have been shown to be crucial in controlling infection caused by the other human gammaherpesvirus, i.e., Epstein-Barr virus (EBV) (11, 14). In support of this hypothesis, our laboratory (40-42) and others (4-6, 12, 19, 23, 26-28, 31, 32, 36, 37, 43, 44) have shown that CD8⁺ T cells produce gamma interferon (IFN-γ) in response to HHV-8 immunodominant epitopes presented by major histocompatibility complex class I (MHC-I) in HHV-8-seropositive individuals. Little is known whether T cells produce other immune mediators in response to HHV-8 infection. Indeed, polyfunctional T cells, i.e., single cells producing two or more immune mediators, have been linked to control of HIV and other persistent infections (1, 24, 29, 33) and could play a role in controlling HHV-8 infection. In one recent study, HHV-8 epitope-specific, polyfunctional T cells were detected in patients with multicentric Castleman''s disease, but these cells did not differ in number from those in healthy controls (13). Another study has found that patients with controlled KS had HHV-8-specific CD8⁺ T cells that secreted IFN-γ and tumor necrosis factor alpha (TNF-α) but that patients with progressive disease had weaker and less polyfunctional CD8⁺ T cells (2).HHV-8 epitope-specific monofunctional and polyfunctional T cell immunity could be important in development of HHV-8 vaccines that induce T cell responses that target these viral epitopes. In the present study, we therefore investigated CD8⁺ T cell responses to two HHV-8 lytic proteins, gB (glycoprotein B) and K8.1, and two latency proteins, LANA-1 (latency associated nuclear antigen-1) and K12. We previously showed that optimal induction of T cell reactivity to the HHV-8 protein gB required 1 week of stimulation with peptide-loaded, autologous, mature, monocyte-derived dendritic cells (DC) (40). Using this enhanced DC-T cell stimulation system, we now have revealed several new epitopes for these four lytic and latency HHV-8 proteins in healthy HHV-8-seropositive individuals, which induce both monofunctional and polyfunctional CD8⁺ T cells. These regions of HHV-8 could be critical in understanding HHV-8 immunopathogenesis and in vaccine development.     

Development of Whole-Virus Multiplex Luminex-Based Serological Assays for Diagnosis of Infections with Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Homologs in Macaques

Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi''s sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.     

Fifty Percent Tissue Culture Infective Dose Assay for Determining the Titer of Infectious Human Herpesvirus 8

We have developed a human herpesvirus 8 (HHV-8) 50% tissue culture infective dose (TCID₅₀) assay using the T1H6-DC-SIGN cell line. Infection of T1H6-DC-SIGN cells with HHV-8 induces expression of β-galactosidase, which was used to determine TCID₅₀ levels. Validation of TCID₅₀ values was performed by immunofluorescence assay of HHV-8 infection of immature dendritic cells at various TCID₅₀ doses.     

Mapping and Serodiagnostic Application of a Dominant Epitope within the Human Herpesvirus 8 ORF 65-Encoded Protein

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi’s sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.