Investigations on the aetiology of pinching off syndrome in four white-tailed sea eagles (Haliaeetus albicilla) from Germany.

The purpose of this study was to investigate the aetiology of the pinching off syndrome (POS), a generalized feather abnormality affecting free-living nestling of the white-tailed sea eagle (Haliaeetus albicilla) in Europe. For the first time, extensive clinical, haematological, biochemical, virological, bacteriological, nutritional, histopathological, parasitological and electron microscopical examinations were performed on three females and one male suffering from POS. Early and increased cytokeratin formation at the base of regenerating feathers and their follicle was observed in affected birds. Ultrathin sections of the feather papillae revealed an extended stratum transitivum and a compact, thickened keratinized stratum corneum. The transitional cells in POS feathers contained vacuoles often associated with the nucleus. Lipofuscin accumulations in neurons, glial cells and islet cells of the pancreas were found in all examined birds. It was not clear whether there is an association between the occurrence of lipofuscin and POS. No evidence was found to suggest that infectious agents (parasites, bacteria, fungi or viruses), malnutrition or hormonal imbalances are involved in the aetiology of POS in white-tailed sea eagles. It remains unclear whether there is a genetic background of POS.

The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis.

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet-activating factor, and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha, promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.

Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer-PCR-based search for a human circovirus.

BACKGROUND: An important issue with respect to virus safety in xenotransplantation is the search for human analogues of porcine viruses, because transmission of a porcine virus followed by recombination with a related human virus may lead to a new emerging virus of unknown pathogenicity, host range and virulence. In case of circoviruses, two types of porcine circovirus (PCV1 and PCV2) are described, but the existence of an analogous human circovirus has not yet been investigated. METHODS: This study describes the analysis of human samples with a consensus primer-PCR approach designed to amplify conserved regions from the rep gene of circoviruses from the genus Circovirus. DNA from human sera, lymph nodes, blood and urine was extracted and investigated with this method that has led previously to the identification of a new avian circovirus. RESULTS: By screening 1101 samples (there of 168 from immunocompromised patients), no evidence for the existence of a human circovirus related to the genus Circovirus was obtained. CONCLUSIONS: This result renders the existence of a human circovirus related to the porcine circoviruses more unlikely, nevertheless the presence of such a virus cannot be ruled out.

S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.

Infection studies on human cell lines with porcine circovirus type 1 and porcine circovirus type 2.

BACKGROUND: The lack of human donor organs in allotransplantation has led to a proposal for the use of porcine tissues and organs as alternative therapeutic material for humans. Besides immunological problems like graft rejection, one of the major concerns is the transmission of porcine microorganisms as viruses, bacteria and fungi to a human recipient. METHODS: Human cell lines have been infected with porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) to investigate whether PCV can infect and replicate in human epithelial cells and lymphocytes. Infection of PCV1 was observed with 293, Hela and Chang liver cells, infection with PCV2 only in Rd cells. In addition, religated viral DNA of PCV1 and PCV2 has been used to transfect adherent human cell lines. RESULTS: PCV1 persisted in most cell lines without causing any visible changes, while PCV2-transfected cells showed a cytopathogenic effect. Presence of PCV DNA was detected in cells and supernatant by PCR, expression of viral proteins by an indirect immune fluorescence assay. A replication assay showed that the replication of PCV DNA was initiated at the origin of replication. When virus-free cells were inoculated with the supernatant of PCV-infected human cells, the infection was not passed. CONCLUSION: Although PCV gene expression and replication took place in human cells, the infection is non-productive. Alteration of protein localization suggests that protein targeting may be disturbed in human cells.

Molecular biology of Porcine circovirus: analyses of gene expression and viral replication.

The rep gene of Porcine circovirus type 1 directs the synthesis of two proteins. The full-length protein Rep is 312 amino acids in size, the spliced variant Rep' is truncated (168 aa) and exon 2 is frame-shifted. Replication of PCV1 DNA depends on synthesis of both proteins. Rep and Rep' bind in vitro to double-stranded DNA fragments comprising part of the origin of replication of PCV1, but the minimal binding sites of the two proteins are distinct. Rep protein represses the promoter of the rep gene by binding to the two inner hexamers H1 and H2. Although Rep' binds to the same sequence, it does not influence Prep. Twelve hours after PCV1 infection, similar amounts of rep and rep' were detected by real-time PCR, but later on, the ratio of the two transcripts varied. Both proteins are co-localised in the nucleus and formation of homo- and heteromeric complexes has been observed. When a replication assay was performed, in which Rep and Rep' protein of PCV1 was used to replicate the origin of PCV1 and PCV2, the rep gene products were found to initiate replication at both origins of replication.

A method to diagnose Pigeon circovirus infection in vivo.

This study describes a method to detect in vivo Pigeon circovirus (PiCV, also called Columbid circovirus, CoCV) identified recently. Blood samples of healthy and diseased pigeons were investigated. DNA was isolated from a drop of blood spotted and dried on filter paper. A nested PCR using two primer pairs from the cap gene was performed to detect PiCV. Using this method, PiCV nucleic acid was detected in 17 of 53 pigeons examined. PCR using DNA from dried blood samples is a simple, fast and reliable technique that allows detection of PiCV in vivo and facilitates collection, storage and shipment of samples.