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 ß-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.

Human herpesvirus 8 infects and replicates in primary cultures of activated B lymphocytes through DC-SIGN.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.

Human herpesvirus 8 glycoprotein B binds the entry receptor DC-SIGN.

We have previously shown that human herpesvirus 8 (HHV-8) uses DC-SIGN as an entry receptor for dendritic cells, macrophages and B cells. The viral attachment protein for DC-SIGN is unknown. HHV-8 virions contain five conserved herpesvirus glycoproteins, a single unique glycoprotein, and two predicted glycoproteins. Previous studies have shown that DC-SIGN binds highly mannosylated glycoproteins. The HHV-8 glycoprotein B (gB) has been reported to be highly mannosylated, and therefore we hypothesized that gB will bind to DC-SIGN. In this report we confirm that gB has a high mannose carbohydrate structure and demonstrate for the first time that it binds DC-SIGN in a dose-dependent manner. We also identify key amino acids in the DC-SIGN carbohydrate recognition domain that are required for HHV-8 infection and compare these results with published binding regions for ICAM-2/3 and HIV-1 gp120. These results clarify some of the initial events in HHV-8 entry and can be used for the design of targeted preventive therapies.

Cytokine production by human herpesvirus 8-infected dendritic cells.

We have shown previously that human herpesvirus 8 (HHV-8)-infected dendritic cells (DCs) undergo incomplete maturation and have a defective antigen-presenting function. Here, we examined the effects of HHV-8 infection on cytokine production, which is critical to the function of DCs. We detected expression of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES and IL-12p40 from 2 to 6 h post-infection, and these peaked by 15-24 h. Expression of these factors decreased 24-48 h post-infection, with the exception of TNF-alpha which remained high throughout the entire 72 h. Interestingly, while IL-12p40 expression increased post-infection, bioactive IL-12p70 was not detected in the supernatants. These results suggest an intentional skewing of cytokine production in HHV-8-infected DCs towards induction of a Th2 response.

Validation of a low cost computer-based method for quantification of immunohistochemistry-stained sections.

The aim of the present study was to determine if the Alpha DigiDoc RT system would be an effective method of quantifying immunohistochemical staining as compared with a manual counting method, which is considered the gold standard. Two readers were used to count 31 samples by both methods. The results obtained using the Bland-Altman for concordance deemed no statistical difference between the 2 methods. Thus, the Alpha DigiDoc RT system is an effective, low cost method to quantify immunohistochemical data.

DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.