Detection of antibodies to Kaposi's sarcoma-associated herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA.

Detection of antibodies to Kaposi's sarcoma-associated herpesvirus (KSHV or Human herpesvirus 8) is a topic of ongoing controversy. KSHV expresses multiple antigens and host responses are highly variable. We have previously described an algorithm for determining KSHV infection based on K8.1 ELISA and LANA immunofluorescence assay (IFA). Here we describe the development of a recombinant ELISA for LANA and an improved testing strategy using ELISAs for LANA and K8.1. We assessed mammalian and baculovirus expression systems for the production of full-length recombinant LANA. We evaluated the performance of LANA ELISAs using human serum samples from several sources including blood donors and clinical patients diagnosed with Kaposi's sarcoma and compared them to LANA IFA. Both LANA ELISAs exhibited comparable sensitivity and specificity to LANA IFA but showed considerably greater reliability. The LANA ELISA can thus be used in conjunction with the previously described K8.1 ELISA to enable the highly sensitive and specific detection of antibodies to KSHV. Use of this testing strategy will provide a more accurate and reliable diagnostic assessment of KSHV status.

Gene regulation networks related to neural differentiation of hESC.

With the unique property of self-renewal and developmental pluripotency, human embryonic stem cells (hESC) provide an opportunity to study molecular aspects of developmental biology. Understanding gene regulation of hESC pluripotency is a critical step toward directing hESC differentiation for regenerative medicine. However, currently little is known about hESC gene regulation of hESC pluripotency. Applying network analysis to microarray gene expression profiling data, we compared gene expression profiles from pluripotent hESC to hESC-derived astrocytes and identified potential gene regulation networks. These gene regulation networks suggest that hECS has stringent control of cell cycle and apoptosis. Our data reveal several potential hESC differentiation biomarkers and suggest that IGF2 and A2M could play a role in hESC pluripotency by altering the availability of cytokines at the local environment of hECS. These findings underscore the importance of network analysis among differentially expressed genes, and should facilitate future study for understanding the gene regulation of hESC pluripotency.

Risk factors for human herpesvirus 8 infection among adults in the United States and evidence for sexual transmission.

BACKGROUND: Human herpesvirus 8 (HHV-8) causes Kaposi sarcoma. In the United States, transmission routes for HHV-8 are uncertain. METHODS: The National Health and Nutrition Examination Survey III sampled individuals from the US general population (1988-1994). We used enzyme immunoassays (EIAs) to measure HHV-8 antibodies (K8.1 and open reading frame [ORF] 73 antigens) in 13,894 surveyed adults. HHV-8 seroprevalence was examined according to sexual history and viral coinfection markers. RESULTS: Overall, seroprevalence was low when a highly specific cutoff was used (K8.1, 1.6%; ORF73, 1.5%) but was higher when a less-specific cutoff was used (K8.1, 7.1%; ORF73, 7.4%). When the more-specific approach was used, K8.1 seroprevalence was similar in men and women. Men who have sex with men (MSM) had a higher K8.1 seroprevalence (8.2%). Among other men, K8.1 seroprevalence was marginally associated with duration of heterosexual activity (P=.1) and was positively associated with the lifetime number of sex partners (P=.04) and with coinfections with hepatitis B virus (6.1% vs. 1.2% without coinfection; P<.001) and herpes simplex virus 2 (2.7% vs. 1.0%; P=.003). Among women, K8.1 seroprevalence was not significantly related to duration of sexual activity, the lifetime number of sex partners, or viral coinfections. The ORF73 EIA revealed similar but less clear-cut patterns. CONCLUSIONS: Among men, HHV-8 transmission may occur through sexual activity, particularly sex with other men. No evidence was observed for heterosexual transmission to women.

Virologic, hematologic, and immunologic risk factors for classic Kaposi sarcoma.

BACKGROUND: Classic Kaposi sarcoma (CKS) is an inflammatory-mediated neoplasm that develops in the presence of KS-associated herpesvirus (KSHV) and immune perturbation. In the current study, the authors compared CKS cases with age-matched and sex-matched KSHV-seropositive controls without human immunodeficiency virus-1 infection and markers of viral control, blood counts, CD4-positive and CD8-positive lymphocytes, and serum beta-2-microglobulin and neopterin levels. METHODS: Viral loads were detected using real-time amplification of the KSHV-K6 and EBV-pol genes, anti-K8.1 (lytic) titers were detected by enzyme-linked immunoadsorbent assay, and antilatent nuclear antigen (LANA) titers were detected using immunofluorescence. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using logistic regression adjusted for sex, age, and study site. RESULTS: Peripheral blood mononuclear cells (PBMC) KSHV DNA detection (P < or = .0001) and high KSHV lytic (>1:1745; P < or = .0001) and latent (>1:102,400; P = .03) antibody titers were found to be positively associated with CKS risk. Antibody titers were higher in cases with lesions compared with cases without lesions (P < or =.05). The detection of Epstein-Barr virus (EBV) DNA in PBMCs was not found to be associated with CKS (P = .95). Independent of PBMC KSHV DNA, CKS risk was found to be positively associated with reduced hematocrit (<37.4%; P = .03), hemoglobin (<12g/dL; P = .04), and lymphocytes (<1000 cells/microL; P = .004), including CD4-positive (+) cells (<457 cells/microL; P = .07) and CD8+ cells (<213cells/microL; P = .04), and with increased monocytes (> or =638 cells/microL; P = .009). Nonsignificant elevations of beta-2-microglobulin and neopterin were observed among cases regardless of disease burden (P > or = .08). In a multivariate model, the CKS risk was found to be associated with PBMC KSHV DNA (OR of 2.7; 95% CI, 1.4-5.3), a high KSHV lytic antibody titer (OR of 3.7; 95% CI, 1.9-7.4), and low lymphocytes, particularly among those patients age <70 years (OR of 8.0; 95% CI, 2.7-23.7). CONCLUSIONS: The findings of the current study appear to corroborate the specificity of KSHV and highlight the hematologic and immunologic correlates involved in the pathogenesis of CKS.

Water, socioeconomic factors, and human herpesvirus 8 infection in Ugandan children and their mothers.

BACKGROUND: Human herpesvirus 8 (HHV-8) infection is common in sub-Saharan Africa, but its distribution is uneven. Transmission occurs during childhood within families by unclear routes. METHODS: We evaluated 600 Ugandan children with sickle cell disease and their mothers for factors associated with HHV-8 seropositivity in a cross-sectional study. HHV-8 serostatus was determined using an HHV-8 K8.1 glycoprotein enzyme immunoassay. Odds ratios for seropositivity were estimated using logistic regression, and factor analysis was used to identify clustering among socioeconomic variables. RESULTS: One hundred seventeen (21%) of 561 children and 166 (34%) of 485 mothers with definite HHV-8 serostatus were seropositive. For children, seropositivity was associated with age, mother's HHV-8 serostatus (especially for children aged 6 years or younger), lower maternal education level, mother's income, and low-status father's occupation (P < 0.05 for all). Using communal standpipe or using surface water sources were both associated with seropositivity (OR 2.70, 95% CI 0.80-9.06 and 4.02, 95% CI 1.18-13.7, respectively) as compared to using private tap water. These associations remained, albeit attenuated, after adjusting for maternal education and child's age (P = 0.08). In factor analysis, low scores on environmental and family factors, which captured household and parental characteristics, respectively, were positively associated with seropositivity (P(trend) < 0.05 for both). For mothers, HHV-8 seropositivity was significantly associated with water source and maternal income. CONCLUSIONS: HHV-8 infection in Ugandan children was associated with lower socioeconomic status and using surface water. Households with limited access to water may have less hygienic practices that increase risk for HHV-8 infection.

Genotypic characterization of Kaposi's sarcoma-associated herpesvirus in asymptomatic infected subjects from isolated populations.

Molecular epidemiological studies of Kaposi's sarcoma-associated herpesvirus (KSHV) have concentrated on characterization of viral strains in tumour biopsy samples from Kaposi's sarcoma (KS) patients, mostly obtained in the United States and Europe. Tumour biopsies are a convenient source of viral DNA, as they have a high viral load compared to peripheral blood. However, sequences obtained from biopsies may not be representative of viral strains in asymptomatic subjects and information on ethnicity is often not available. Here, a population-based approach has been used to study the molecular and seroepidemiology of KSHV in isolated populations in Ecuador and Botswana. Amerindians in Ecuador had a variable prevalence of KSHV and all strains characterized were of subtype E, based on K1 sequencing. All Amerindian strains had predominant (P)-type K15 alleles and had sequences in both T0.7 and ORF 75 that appeared to be characteristic of these strains. The prevalence of KSHV in two ethnic groups in Botswana was extremely high. K1 sequences from both Bantu and San subjects were mostly of subtypes B and A5, which are typical of African KSHV strains, but the sequence from one San subject did not cluster with any known subtype. Considerable heterogeneity was seen in the T0.7 and ORF 75 genes in the San subjects and one had a minor (M)-type K15 allele. The heterogeneity of the KSHV strains found in these subjects from Botswana contrasts with the homogeneity of KSHV strains in Amerindians, reflecting differences in the evolutionary history of these populations.

Detection and quantification of Kaposi's sarcoma-associated herpesvirus to predict AIDS-associated Kaposi's sarcoma.

OBJECTIVE: To identify immunologic and virologic predictors of AIDS-associated Kaposi's sarcoma (KS). DESIGN: Nested case-control analysis of KS risk in a cohort of 132 HIV-infected homosexual men in New York and Washington, DC, USA. METHODS: For each KS case, we selected two HIV-infected controls, matched for CD4 cell count and Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8; KSHV) serostatus (enzyme immunoassay for antibody to KSHV protein K8.1). Cell-associated KSHV and Epstein-Barr virus (EBV) viral loads were measured with quantitative real-time PCR assays on samples collected 1 year (median) before KS diagnosis. RESULTS: Thirty-one men developed AIDS-associated KS (incidence 3.1 per 100 person years). Among HIV-infected men, KS incidence was higher among those with K8.1 seropositivity (5.0 versus 1.4 per 100 person years; P = 0.004), low CD4 cell count [hazard ratio (HR), 1.49; 95% confidence interval (CI), 1.24-1.79 per 100 x 10(6) cells/l decline), or high HIV RNA level (HR, 3.96; 95% CI, 2.19-7.16 per log(10)). In the case-control analysis, nine of 70 evaluated subjects had KSHV viremia, generally low level (median viral load 180 copies per 1 x 10(6) cells). KSHV viremia was associated with increased KS risk (unadjusted odds ratio, 9.1; 95% CI, 1.7-48; odds ratio, 11.7; 95% CI, 1.8-76 after adjustment for K8.1 serostatus, CD4 cell count, and HIV RNA). Among K8.1-seropositive subjects, KS incidence was tenfold higher in those with KSHV viremia (30.3 per 100 person years versus 3.4 per 100 person years in those without viremia). Also, EBV viral loads were higher in cases than in controls (P = 0.07). CONCLUSIONS: Among individuals with HIV-KSHV coinfection, KSHV viremia identifies a subgroup with extremely high risk for developing KS.

Novel Kaposi's sarcoma-associated herpesvirus homolog in baboons.

Kaposi's sarcoma (KS) and lymphoproliferative diseases induced by KS-associated herpesvirus (KSHV/human herpesvirus 8) cause substantial morbidity and mortality in human immunodeficiency virus-infected individuals. To understand KSHV biology it is useful to investigate closely related rhadinoviruses naturally occurring in nonhuman primates. Here we report evidence for a novel KSHV homolog in captive baboon species (Papio anubis and other). Using degenerate PCR we identified a novel rhadinovirus, PapRV2, that has substantial sequence identity to two essential KSHV genes, the viral polymerase and thymidylate synthase. A subset of animals exhibited detectable PapRV2 viral load in peripheral blood mononuclear cells. Extensive serological analysis of nearly 200 animals in the colony demonstrated that the majority carried cross-reacting antibodies that recognize KSHV or macaque rhadinovirus antigens. Seroreactivity increased with age, similar to the age-specific prevalence of KSHV in the human population. This establishes baboons as a novel resource to investigate rhadinovirus biology, which can be developed into an animal model system for KSHV-associated human diseases, vaccine development, and therapy evaluation.

Seroprevalence of human herpesvirus 8 among injection drug users in San Francisco.

The association between injection drug use and human herpesvirus 8 (HHV-8) was examined to investigate bloodborne transmission of the virus. In all, 1905 injection drug users (IDUs) enrolled in a cross-sectional study were tested for K8.1 antibodies to HHV-8 lytic antigen. Logistic regression was used to adjust for demographic and sexual behavior variables. HHV-8 seroprevalence was 10% among women, 10% among heterosexual men, and 23% among men who have sex with men. In adjusted analyses, HHV-8 seroprevalence increased with longer duration of injection drug use for each of these groups (P = .01, P = .03, and P = .049 for trend, respectively). HHV-8 infection is relatively common among IDUs in San Francisco, and longer duration of injection drug use is associated with an increase in the risk of HHV-8 infection that is not explained by sexual behavior or demographic differences. These results are consistent with the occurrence of bloodborne transmission of HHV-8 among IDUs.