Intrafamiliar transmission of Kaposi's sarcoma-associated herpesvirus and seronegative infection in family members of classic Kaposi's sarcoma patients.

The link between Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) and Kaposi's sarcoma has been proven, but the transmission routes, especially in the heterosexual population, are not yet completely understood. To assess the intrafamilial patterns of transmission among first-degree relatives of Italian classic Kaposi's sarcoma (cKS) patients, KSHV seroprevalence and the presence of viral DNA in blood and saliva were evaluated in 18 families (32 cKS patients and 35 family members), comparing the results with those obtained in 200 elderly healthy controls without known exposure to KSHV. The KSHV genotype of variable region VR1 of the hypervariable ORF K1 gene was subsequently analysed in all KSHV-positive samples. The results showed that KSHV infection was significantly higher in relatives of cKS patients (11/35 cases) than in healthy controls (17/200 cases; P=0.001). The 11 infected relatives included spouses (n=3), siblings (n=2) and offspring (n=6) of the cKS patients; the same KSHV genotype was shared within the same family in the majority of cases (85%), indicating the presence of person-to-person transmission within families. Viral DNA was mostly observed in the saliva of infected relatives (45.4%); detection of DNA in blood was less frequent (27.3%). Notably, KSHV DNA was present in saliva and/or blood of three KSHV-infected relatives with indeterminate or negative serostatus. Thus, the risk of KSHV infection is greatly enhanced within families of cKS patients, where close contacts (horizontal and/or sexual) can contribute to the spread of KSHV.

HHV8 a subtype is associated with rapidly evolving classic Kaposi's sarcoma.

The link between human herpesvirus 8 (KSHV) and Kaposi's sarcoma (KS) has been proven, but many important aspects including risk factors, genetic predisposition to tumor development, transmission of KSHV, and the pathogenic potential of different genotypes remain to be elucidated. Possible associations between clinical parameters and antibody levels, viral load fluctuations, and viral genotype were analyzed by quantitative real-time PCR, an in-house developed IFA assay, and sequence analysis of ORF K1-VR1 in blood, serum and saliva of 38 subjects with classic KS (cKS). KSHV lytic antibodies were significantly increased in stage IV compared to stage I and II patients (p = 0.006 and p = 0.041, respectively). KSHV blood, serum, and saliva viral load was comparable in all stages. The highest viral loads were detected in saliva, and they decreased in stages III-IV compared to stages I-II patients. Higher concentrations of lytic antibodies and higher viral loads were observed in fast progressing cKS patients, in whom KSHV detection from blood was also more frequent. Type A KSHV strain was almost exclusively present in rapid progressors (12/17 cases), while C type was mainly present in slow progressing patients (6/7 cases). Finally, detection of type A KSHV strain associated with higher blood viral loads. KSHV lytic antibody levels and viral load can be used to monitor clinical evolution of cKS. Infection supported by KSHV A subtype is associated with more rapid progressive disease. Careful monitoring and aggressive therapeutic protocols should be considered in patients with KSHV A-supported infection.

Big endothelin-1 and interleukin-6 modulation in human microvascular endothelial cells after human herpesvirus 8 infection.

Endothelin (ET)-1 is an angiogenic factor that, among others, is secreted by endothelial cells during development of several neoplasias. In particular, Kaposi sarcoma (KS) skin lesions show overexpression of the ET-1 system. Spindle cells, which characterize tumor lesions, are of endothelial origin and during disease are infected by human herpesvirus 8 (HHV-8). The majority of these cells are latently infected, suggesting that latent genes are sufficient for maintenance of viral infection and development of KS. The establishment of a reliable infection system is required to better understand the role of viral and cellular angiogenic factors involved in KS progression. For this purpose, we used human microvascular endothelial cells (HMEC-1) to establish an ET-1-producing model of infection with HHV-8. Viral particles purified from BCBL-1 cells were used to infect HMEC-1 monolayer, and infection was assessed by polymerase chain reaction, reverse transcription polymerase chain reaction, and confocal microscopy. Mitochondrial activity and cell viability, measured at 24, 48, and 72 hours after infection by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was reduced in HHV-8-infected cells compared with control. In contrast, 1 week after infection, HHV-8-positive cells showed higher mitochondrial functionality. Endothelin production was measured in culture media collected at 24, 48, and 72 hours after infection. The levels of endothelin precursor big endothelin-1 was increased 3 days after infection, although big ET-1 and ET-1 production did not differ significantly between infected and uninfected cells. These results indicate this model as a useful tool to further characterize the effects of HHV-8 in the early and late phases of infection, and to determine its ability to interfere with the endothelin system.

Human polyomavirus JCV late leader peptide region contains important regulatory elements.

Transcription is a complex process that relies on the cooperative interaction between sequence-specific factors and the basal transcription machinery. The strength of a promoter depends on upstream or downstream cis-acting DNA elements, which bind transcription factors. In this study, we investigated whether DNA elements located downstream of the JCV late promoter, encompassing the late leader peptide region, which encodes agnoprotein, play regulatory roles in the JCV lytic cycle. For this purpose, the entire coding region of the leader peptide was deleted and the functional consequences of this deletion were analyzed. We found that viral gene expression and replication were drastically reduced. Gene expression also decreased from a leader peptide point mutant but to a lesser extent. This suggested that the leader peptide region of JCV might contain critical cis-acting DNA elements to which transcription factors bind and regulate viral gene expression and replication. We analyzed the entire coding region of the late leader peptide by a footprinting assay and identified three major regions (region I, II and III) that were protected by nuclear proteins. Further investigation of the first two protected regions by band shift assays revealed a new band that appeared in new infection cycles, suggesting that viral infection induces new factors that interact with the late leader peptide region of JCV. Analysis of the effect of the leader peptide region on the promoter activity of JCV by transfection assays demonstrated that this region has a positive and negative effect on the large T antigen (LT-Ag)-mediated activation of the viral early and late promoters, respectively. Furthermore, a partial deletion analysis of the leader peptide region encompassing the protected regions I and II demonstrated a significant down-regulation of viral gene expression and replication. More importantly, these results were similar to that obtained from a complete deletion of the late leader peptide region, indicating the critical importance of these two protected regions in JCV regulation. Altogether, these findings suggest that the late leader peptide region contains important regulatory elements to which transcription factors bind and contribute to the JCV gene regulation and replication.

Association of HLA-DRB1 and -DQB1 with Classic Kaposi's Sarcoma in Mainland Italy.

Classic Kaposi's sarcoma (CKS) is a multifocal vascular mesenchymal tumour of unknown origin. Human herpesvirus 8 (HHV8) is now considered to be strongly involved, as a necessary co-factor, though insufficient for development of the disease. Additional identified risk factors include environmental factors, personal habits and genetic susceptibility, with different loci suspected of being risk factors for CKS. Since various human leukocyte antigen (HLA) patterns have been suggested as potential host-related co-factors, the distribution of these alleles was studied in 41 CKS patients, 285 geographically-matched healthy controls (HC) and 17 HHV8-positive controls. Molecular typing of HLA was performed using the polymerase chain reaction sequence-specific primer method (SSP-PCR). Frequency distribution was evaluated by the Chi-squared test with Yates' correction. Odds ratios (OR) and respective 95% confidence limits (CI) were calculated. A significantly higher frequency of HLA-DRB1*13 was observed among the CKS patients (20.7%) compared to the HC (9.8%) (p<0.01; OR: 2.32; 95%CI: 1.21-4.41). Overall, these results indicated that HLA-DRB1*13 may play a role in the development of CKS, while HLA-DQB1*0604 allele involvement occurs in linkage disequilibrium with HLA-DRB1*13. To our knowledge, this is the first study documenting an HLA-DRB1 and -DQB1 loci association with CKS development in the mainland Italian population.

Members of the AP-1 family, c-Jun and c-Fos, functionally interact with JC virus early regulatory protein large T antigen.

The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.

Long-term outcome (35 years) of hepatitis C after acquisition of infection through mini transfusions of blood given at birth.

Long-term follow up studies of hepatitis C virus (HCV) infection rarely exceed 20-25 yr. We studied the outcome of HCV infection in 35-yr-old adults infected at birth (1968) through mini transfusions of blood. A retrospective-prospective study was carried out. The cohort included 31 individuals who were given mini blood transfusions (21-30 ml) collected from a donor subsequently revealed to be HCV infected. At enrollment (1998), 18 of 31 (58.1%) recipients had anti-HCV antibody and 16 (88.9%) of them were HCV-RNA positive. All viremic recipients and the infectious donor had the same genotype 1b. Sequence analysis of E1/E2 and NS5b regions, coupled with phylogenetic analysis, indicated that HCV isolates from donor/recipients were linked. Eleven of the 16 viremic recipients gave consent to liver biopsy. Nine had no fibrosis or mild portal fibrosis and 2 had either discrete (Ishak's staging 3) or marked (Ishak's staging 4) fibrosis. During the prospective follow-up period (1998-2003), 2 patients were given therapy, one of whom achieved sustained clinical and virologic response. A second biopsy, performed in 5 patients at a 5 yr interval, revealed no substantial modifications in 4 cases and progression from absence of fibrosis to mild portal fibrosis in the fifth. In conclusion, taking into account the limited study sample, these findings suggest that HCV infection acquired early in life shows a slow progression and mild outcome during the first 35 yr of infection.