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.     

Novel monoclonal antibodies for identification of multicentric Castleman's disease; Kaposi's sarcoma-associated herpesvirus-encoded vMIP-I and vMIP-II

Recent studies have indicated that vMIP-I and vMIP-II play important roles in the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV)-related diseases due to the effects of these proteins on vascularization. We developed monoclonal antibodies against KSHV-encoded viral macrophage inflammatory protein-I (vMIP-I) and vMIP-II to study these expression profiles and reveal the pathogenesis of KSHV-related diseases. The MAbs against vMIP-I and vMIP-II reacted to KSHV-infected cell lines after lytic induction. Both vMIP-I and the vMIP-II gene products were detected 24 h post-induction with 12-O-tetradecanoylphorbol-13-acetate until 60 h in the cytoplasm of primary effusion lymphoma cell lines. In clinical specimens, both vMIP-I and vMIP-II gene products were detected in the tissues of patients with multicentric Castleman's disease. On the other hand, only vMIP-II was detected in a subset of Kaposi's sarcoma. We concluded that these antibodies might be powerful tools to elucidate the pathogenesis of KSHV-related diseases.     

Human herpesvirus-8: detection of novel herpesvirus-like DNA sequences in Kaposi's sarcoma and other lesions

Kaposi's sarcoma (KS) is a malignancy suspected of having an infectious etiology. Unique viral DNA sequences were recognized in KS lesions, using a novel technique that identifies small differences between two complex genomes. The virus had homology with the herpesvirus family, especially Epstein Barr virus (EBV), yet it was distinct from the known herpesviridae, and was appropriately named human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV). HHV-8 DNA sequences were present in AIDS-associated KS, classic KS, African endemic KS, Mediterranean KS, iatrogenic KS, and KS in homosexual men without HIV infection. HHV-8 DNA sequences were also present in peripheral blood mononuclear cells (PBMC) of KS+ patients; body-cavity-based lymphomas in HIV positive patients without KS; and in tissue from a number of malignant and non-malignant lesions in patients without HIV infection. The role of HHV-8 in KS and other malignancies is not known. Viruses are notoriously trophic for lesional tissue. Therefore, in order to determine the role of HHV-8 in KS pathogenesis, HHV-8 needs to be isolated and shown to induce immortalization in a suitable system. Regardless of its role in KS, another human herpesvirus has been discovered, and the extent of its pathogenicity needs to be uncovered.Abbreviations KS Kaposi's sarcoma - HHV-8 human herpesvirus-8 - KSHV Kaposi's sarcoma-associated herpesvirus - EBV Epstein-Barr virus RDA representational difference analysis     

Quantification of human herpesvirus 8 by real-time PCR in blood fractions of AIDS patients with Kaposi's sarcoma and multicentric Castleman's disease

Few studies have assessed human herpesvirus 8 (HHV8) viremia levels in different HHV8-related pathologies, using sensitive and reproducible molecular assays. Our objective was to compare the HHV8 DNA load in serial blood samples (collected every 3 months for 1 year) from acquired immunodeficiency syndrome (AIDS) patients with Kaposi's sarcoma (KS) and multicentric Castleman's disease (MCD). The HHV8 viral load was determined in both peripheral blood mononuclear cells (PBMC) and plasma fractions, using a competitive real-time polymerase chain reaction (PCR) assay developed in a LightCycler instrument (Roche Diagnostics). In six subjects with limited or extensive KS while on highly active antiretroviral therapy, the HHV8 DNA load was either undetectable (<50 copies/10(5) cells) or low (1,000 copies in at least one of the samples from the two subjects with both KS and MCD. HHV8 DNA was detected in plasma only when the cellular viral load was >10,000 copies/10(5) cells. After chemotherapy, the HHV8 DNA load became undetectable in the MCD patients despite no changes in CD4 T-cell counts or highly active antiretroviral therapy (HAART) regimens. These results suggest that the pathogenesis of the two HHV8-associated diseases (i.e., KS and MCD) might be different, as only the latter was associated with important viremia in our patients.     

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.     

Expression of Kaposi's sarcoma-associated herpesvirus-encoded K10/10.1 protein in tissues and its interaction with poly(A)-binding protein

The K10/10.1 protein is encoded by a cluster of interferon regulatory factor (IRF) homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8, HHV-8) genome. In the present study, we showed that an anti-K10 antibody reacted with a 110-kDa protein encoded by the K10/10.1 gene of KSHV in KSHV-infected primary effusion lymphoma (PEL) cell lines. Expression of K10/10.1 protein was induced by phorbol ester in KSHV-infected cells. A reporter gene assay demonstrated that K10/10.1 protein did not influence promoter activity of human interferon genes, regardless of its homology to human IRFs. Poly(A)-binding protein (PABP) was identified as a partner of K10/10.1 protein. Immunoprecipitation revealed that K10/10.1 protein interacted with PABP specifically in PEL cell lines. IFA revealed co-localization of K10/10.1 protein and PABP in the nucleus of KSHV-infected cells. These data suggest that K10/10.1 protein may affect the translational status or stability of mRNA in host cells.     

Vironome of Kaposi sarcoma associated herpesvirus-inflammatory cytokine syndrome in an AIDS patient reveals co-infection of human herpesvirus 8 and human herpesvirus 6A

KSHV inflammatory cytokine syndrome (KICS) is a newly described condition characterized by systemic illness as a result of systemic, lytic KSHV infection. We used Illumina sequencing to establish the DNA vironome of blood from such a patient. It identified concurrent high-level viremia of human herpesvirus (HHV) 8 and 6a. The HHV8 plasma viral load was 5,300,000 copies/ml, which is the highest reported to date; this despite less than five skin lesions and no HHV8 associated lymphoma. This is the first report of systemic HHV6a/KSHV co-infection in a patient. It is the first whole genome KSHV sequence to be determined directly from patient plasma rather than cultured or biopsied tumor material. This case supports KICS as a new clinical entity associated with KSHV.