A functional toll-like receptor 8 variant is associated with HIV disease restriction.

BACKGROUND: Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS: We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS: This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.

Endoglin expression in metastatic breast cancer cells enhances their invasive phenotype.

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-beta (TGF-beta). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-beta signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-beta. In 3D spheroid confrontation assays between breast tumor cells and TGF-beta-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-beta antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

Susceptibility of different eukaryotic cell lines to SARS-coronavirus.

In order to define and characterize target cells of SARS-coronavirus (SARS-CoV) we studied the susceptibility of 23 different permanent and primary eukaryotic cell lines to SARS-coronavirus. Beneath Vero E6 cells SARS- Coronavirus infection could also be demonstrated in two pig cell lines (POEK, PS) and one human cell line (Huh-7) using the indirect immunofluorescence assay and a newly established quantitative real-time PCR. In all susceptible cell lines mRNA of the Angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV infection, could be detected by RT-PCR. Our results show that there is a correlation between the abundance of ACE2 mRNA and SARS-CoV susceptibility.

In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2.

The aim of this study was to describe the generation of a PCV2 (porcine circovirus type 2) infectious clone (pIC-PCV2) and its infectivity under in vitro and in vivo conditions. The constructed pIC-PCV2 contained the whole PCV2 genome from a German isolate together with a partial duplication of 467 bp. PK-15 cells were transfected with pIC-PCV2 and an indirect immune fluorescence assay (IFA) was performed 7 days post-transfection. The PCV2 Cap gene was expressed in approximately 20 % of the cultured cells, and only the recombination product, and not pIC-PCV2, was subsequently detected by PCR and Southern blot. This result indicated that infection by pIC-PCV2 delivered genomic PCV2 DNA specifically into susceptible cells and led to the expression of a functional virus genome. Eighteen 30- to 40-day-old conventional pigs were distributed into three groups. Group 1 pigs (n=6) were inoculated intranasally (i.n.) with a Spanish isolate of PCV2 propagated in cell culture; pigs from group 2 (n=6) were inoculated with pIC-PCV2 intramuscularly (i.m.), and the last group of pigs (n=6) was inoculated with pIC-PCV2 intraperitoneally (i.p.). All pigs remained clinically healthy during the whole experimental period (35 days). Pigs that received pIC-PCV2 i.p. and i.m., as well as those PCV2 i.n. inoculated, became infected based on an in situ hybridization (ISH), PCR, TaqMan PCR and serological results. The results of this study confirm that cloned PCV2 genomic DNA is infectious both in vitro and in vivo, and is able to cause PMWS-like lesions in i.p. and i.m. experimentally inoculated pigs.

Cloning and sequencing of Duck circovirus (DuCV).

The genome of Duck circovirus (DuCV) is circular and 1996 nts in size. Two major open reading frames were identified, encoding the replicase (V1) and the capsid protein (C1). A stem-loop structure comprising the nonamer 5'-TATTATTAC, conserved in all circo-, nano- and geminiviruses, was found. Unique to DuCV, the region between the 3'-ends of the rep and cap gene contains four repeats of a 44-bp sequence. Phylogenetic analysis shows close relation of DuCV with Goose circovirus and suggests classification of DuCV as a new member of the genus Circovirus of the virus family Circoviridae.

A diagnostic study on columbid circovirus infection.

A systematic study was performed to examine the frequency of columbid circovirus (CoCV) infection in diseased young pigeons submitted for necropsy and its relevance to pigeon health. Existing diagnostic methods were compared. Among 176 diseased young pigeons examined, CoCV infection was detected in 83 cases using negative contrast electron microscopy. Histopathological examination allowed a clear diagnosis in only 42 pigeons. Therefore, a polymerase chain reaction assay and an in situ hybridization test were developed as additional diagnostic tools. CoCV is by far the most frequently detected infectious agent in diseased young pigeons. Infected pigeons reveal a broad range of concurrent infections. Pathological findings suggest an immunosuppressive effect of CoCV.

Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons.

The complete nucleotide sequence of columbid circovirus (CoCV) isolated from pigeons is described. CoCV was amplified using a consensus primer PCR approach directed against conserved sequences within the rep genes of vertebrate circoviruses. The genome of CoCV is circular and 2037 nt in size. It displays 55% homology to the genome of psittacine beak and feather disease virus and is more distantly related (< 40% homology) to porcine circovirus type 1 and 2. Two major open reading frames were identified, encoding the replicase and the putative capsid protein of CoCV. A region similar to the origin of replication of other circoviruses was found: it encompasses a stem-loop structure with the nonamer 5'-TAGTATTAC, conserved in circo-, nano- and geminiviruses. Phylogenetic analyses suggest classification of CoCV as member of the genus Circovirus of the virus family Circoviridae.