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Peter Staeheli

Publications and source records attributed to Peter Staeheli.

36 records · Page 2Linked to original sources
PubMed2004

Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice.

The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.

Animals↗
PubMed2004

Borna disease virus multiplication in mouse organotypic slice cultures is site-specifically inhibited by gamma interferon but not by interleukin-12.

Borna disease virus (BDV) induces a nonpurulent CD4- and CD8-T-cell-dependent meningoencephalitis in susceptible animals. Upon intracerebral infection, BDV replicates in the mouse central nervous system (CNS), but only a few mouse strains develop neurological disorder. The antiviral T cells appear to suppress BDV replication by a noncytolytic mechanism. Since BDV does not replicate in standard mouse cell cultures, the putative role of gamma interferon (IFN-gamma) in virus control could not be tested experimentally. Here, we report that mouse organotypic slice cultures can be used to elucidate the complex interactions of BDV, the CNS, and the immune system. We show that BDV replicated in various cell types of mouse cerebellar slice cultures in vitro. In infected slice cultures, a moderate upregulation of the chemokine genes CCL5 and CXCL10 was observed, while expression of various neural genes as well as other chemokine and cytokine genes was not altered. IFN-gamma inhibited the multiplication of BDV in cerebellar and hippocampal slice cultures in a dose-dependent manner. However, while complete suppression of BDV was observed in cerebellar slice cultures, inhibition was incomplete in hippocampal slice cultures. Kinetic studies indicated that IFN-gamma protects noninfected cells from infection rather than clearing the virus from infected cells. These results demonstrate that BDV can replicate in cultured neural cells of the mouse if organ integrity is well preserved. They further show that IFN-gamma is a powerful inhibitor of BDV in the absence of blood-borne leukocytes in mouse cerebellar slice cultures.

Animals↗
PubMed2004

Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease.

The nucleoprotein (N) of Borna disease virus (BDV) is the major target of the disease-inducing antiviral CD8 T-cell response in the central nervous system of mice. We established two transgenic mouse lines which express BDV-N in either neurons (Neuro-N) or astrocytes (Astro-N). Despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. When Neuro-N mice were infected as adults, replication of BDV was severely impaired and was restricted to brain areas with a low density of transgene-expressing cells. Notably, the virus failed to replicate in the transgene-expressing granular and pyramidal neurons of the hippocampus (which are usually the preferred host cells of BDV). When Neuro-N mice were infected within the first 5 days of life, replication of BDV was not suppressed in most neurons, presumably because the onset of transgene expression in the brain occurred after these cells became infected with BDV. Astro-N mice remained susceptible to BDV infection, but they were resistant to BDV-induced neurological disorder. Unlike their nontransgenic littermates, Neuro-N mice with persistent BDV infection did not develop neurological disease after immunization with a vaccinia virus vector expressing BDV-N. In contrast to the situation in wild-type mice, this treatment also failed to induce N-specific CD8 T cells in the spleens of both transgenic mouse lines. Thus, while resistance to BDV infection in N-expressing neurons appeared to result from untimely expression of a viral nucleocapsid component, the resistance to BDV-induced neuropathology probably resulted from immunological tolerance.

Animals↗
PubMed2003

IL-18 stimulates the proliferation and IFN-gamma release of CD4+ T cells in the chicken: conservation of a Th1-like system in a nonmammalian species.

The phylogeny of Th1 and Th2 subsets has not been characterized mainly due to the limited information regarding cytokines in nonmammalian vertebrates. In this study, we characterize a Th1-like regulatory system focusing on the IL-18-regulated IFN-gamma secretion. Stimulation of splenocytes with chicken IL-18 induced high levels of IFN-gamma secretion. Depletion of either macrophages or CD4(+) T cells from the splenocyte cultures caused unresponsiveness to IL-18. In contrast, PBL were unresponsive to IL-18 in the presence or absence of macrophages, but IFN-gamma secretion was stimulated by suboptimal anti-TCR cross-linking combined with IL-18. Splenocytes from five different chicken lines responded equally well to the IL-18 treatment. LSL chicken splenocytes, however, responded only to IL-18 when stimulated either with optimal TCR cross-linking alone or suboptimal TCR cross-linking combined with IL-18. IL-18 not only induced IFN-gamma secretion, but also stimulated splenocyte proliferation. This IL-18-induced proliferation was compared with the effects observed with IL-2. Both cytokines activated the splenocytes as demonstrated by increased size and MHC class II Ag up-regulation in the case of IL-18. Phenotypic analyses following 6 days of culture revealed that IL-2 mainly affected the proliferation of CD8(+) cells, whereas IL-18 had an opposite effect and stimulated the proliferation of CD4(+) cells. Taken together, these results demonstrate the conservation of Th1-like proinflammatory responses in the chicken; they characterize IL-18 as a major growth factor of CD4(+) T cells and identify two distinct mechanisms of IL-18-induced IFN-gamma secretion.

Animals↗
PubMed2003

A sensitive bioassay for chicken interleukin-18 based on the inducible release of preformed interferon-gamma.

Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.

Animals↗
PubMed2003

Truncated chicken interleukin-1beta with increased biologic activity.

Chicken interleukin-1beta (ChIL-1beta) is synthesized as a precursor molecule that unlike its mammalian counterpart, lacks a typical caspase-1 cleavage site. Therefore, it was unclear if proteolytic cleavage of ChIL-1beta can occur and if cleavage might modulate the biologic activity of this cytokine. Using an avian indicator cell line that carries an NF-kappaB-regulated luciferase reporter gene, we established a sensitive and highly specific bioassay for ChIL-1beta. Experiments with a rabbit antiserum indicated that the NF-kappaB-stimulating activity in supernatants of lipopolysaccharide (LPS)-treated chicken HD-11 macrophages is largely due to IL-1beta and that proteolytic processing of natural and recombinant ChIL-1beta is not very efficient. Functional analyses further revealed that cDNAs for either full-length or N-terminally truncated chicken ChIL-1beta yielded active cytokine. A truncated molecule that closely resembled putative mature ChIL-1beta exhibited more than 100-fold enhanced biologic activity after expression in mammalian cells, indicating that precursor cleavage is indeed of critical importance for maximal activity.

Animals↗
PubMed2003

Viral evasion of the interferon system: old viruses, new tricks.

Interferon-alpha/beta (IFN-alpha/beta) plays an important role in antiviral defense, but many viruses have evolved mechanisms to evade this barrier. The emerging understanding of how viral proteins intercept the IFN response was the focus of a workshop held at the University of Freiburg, Germany, on September 27-28, 2002.

Animals↗
PubMed2003

Active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein.

Analysis of the composition and regulation of the Borna disease virus (BDV) polymerase complex has so far been limited by the lack of a functional assay. To establish such an assay on the basis of an artificial minigenome, we constructed expression vectors encoding either nucleoprotein (N), phosphoprotein (P), X protein, or polymerase (L) of BDV under the control of the chicken beta-actin promoter. A Flag-tagged version of L colocalized with virus-encoded N and P in characteristic nuclear dots of BDV-infected cells and increased viral N-protein levels in persistently infected Vero cells. Vector-driven expression of L, N, and P in BSR-T7 cells together with a negative-sense BDV minigenome carrying a chloramphenicol acetyltransferase (CAT) reporter gene resulted in efficient synthesis of CAT protein. Induction of CAT protein synthesis strongly depended on a 10- to 30-fold molar excess of the N-encoding plasmid over the P-encoding plasmid. Cotransfection of even small amounts of plasmid encoding the viral X protein reduced CAT synthesis to background levels. Thus, the N-to-P stoichiometry seems to play a central role in the regulation of the BDV polymerase complex. Our data further suggest a negative regulatory function for the X protein of BDV.

Animals↗
PubMed2003

Rat model of borna disease virus transmission: epidemiological implications.

Rapid transmission of Borna disease virus occurred upon cohabitation of persistently infected and naive rats. Infectious virus, which was abundantly present in fresh urine samples of carrier rats, entered the brains of recipient rats via the olfactory route. Thus, susceptible farm animals possibly acquire the virus from persistently infected rats.

Animals↗
PubMed2003

Selective virus resistance conferred by expression of Borna disease virus nucleocapsid components.

Persistent viral infections can render host cells resistant to superinfection with closely related viruses by largely uncharacterized mechanisms. We present evidence for superinfection exclusion in brains of Borna disease virus (BDV)-infected rats and in persistently infected Vero cells, and we suggest that acquired resistance to BDV is due to unbalanced intracellular levels of viral nucleocapsid components. We observed that expression of BDV protein P, N, or X rendered human cells resistant to subsequent challenge with BDV but not with other RNA viruses, indicating that incorrect stoichiometry of nucleocapsid components selectively blocked the polymerase activity of incoming viruses. Vero cells containing high levels of an untranslatable BDV-N transcript remained virus susceptible, demonstrating that viral protein rather than RNA mediated resistance. Transient overexpression of BDV-P in persistently infected Vero cells was also remarkably effective against BDV, indicating that the intracellular balance of viral nucleocapsid components could serve as a target for future therapeutic antiviral strategies.

Animals↗
PubMed2002

An interferon-gamma-binding protein of novel structure encoded by the fowlpox virus.

Poxviruses have evolved various strategies to counteract the host immune response, one of which is based on the expression of soluble cytokine receptors. Using various biological assays, we detected a chicken interferon-gamma (chIFN-gamma)-neutralizing activity in supernatants of fowlpox virus (FPV)-infected cells that could be destroyed by trypsin treatment. Secreted viral proteins were purified by affinity chromatography using matrix-immobilized chIFN-gamma, followed by two-dimensional gel electrophoresis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis indicated that the viral IFN-gamma-binding protein in question was encoded by the FPV gene 016. The chicken IFN-gamma binding and neutralizing activity of the recombinant FPV016 protein was confirmed using supernatants of cells infected with a recombinant vaccinia virus that lacked its own IFN-gamma-binding protein but instead expressed the FPV016 gene. The FPV016 gene product also neutralized the activity of duck and human IFN-gamma but failed to neutralize the activity of mouse and rat IFN-gamma. Unlike previously known cellular and poxviral IFN-gamma receptors, which all contain fibronectin type III domains, the IFN-gamma-binding protein of FPV contains an immunoglobulin domain. Remarkably, it exhibits no significant homology to any known viral or cellular protein. Because IFN-gamma receptors of birds have not yet been characterized at the molecular level, the possibility remains that FPV016 represents a hijacked chicken gene and that avian and mammalian IFN-gamma receptors have fundamentally different primary structures.

Amino Acid Sequence↗
PubMed2002

High-avidity human serum antibodies recognizing linear epitopes of Borna disease virus proteins.

BACKGROUND: The recent observation that Borna disease virus (BDV)-reactive antibodies from psychiatric patients exhibit only low avidity for BDV antigen called into question their diagnostic value and raised the possibility that antigenically related microorganisms or self antigens caused the production of these antibodies. We further characterized the specificity of these antibodies. METHODS: We established a peptide array-based screening test that allows the identification of antibodies directed against linear epitopes of the two major BDV proteins, the nucleoprotein (N) and the phosphoprotein (P). RESULTS: Initial tests employing sera of BDV-infected mice and rats or horses with Borna disease revealed a high specificity and sensitivity of this test. All sera recognized epitopes of N, P, or both. Sera of noninfected rats, mice, and horses showed no signals on either peptide array. Several human sera that recognized BDV antigen by indirect immunofluorescence contained antibodies that recognized various linear epitopes of one or even both BDV proteins. Remarkably, antibodies purified from such human serum by matrix-immobilized peptides showed high-avidity binding to BDV antigens when assayed by IFA or Western blotting. CONCLUSIONS: These data suggest that reactive antibodies found in psychiatric patients indeed indicate infection with BDV or a BDV-like agent. However, the poor affinity maturation of BDV-specific human antibodies remains unexplained.

Animals↗
PubMed2002

Bornaviruses.

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Animals↗
PubMed2002

The effects of thymulin on macrophage responsiveness to interferon-gamma.

The effect of in vivo and in vitro thymulin treatments on macrophage responsiveness to interferon-gamma was evaluated in chickens. Seven-week-old chickens were treated with 0, 1, 10, or 100ng thymulin per 100g body weight. Abdominal exudate cells (AEC), a source of macrophages, were harvested and cultured in the presence of graded levels of recombinant chicken interferon-gamma (ChIFN-gamma). Responsiveness to ChIFN-gamma was determined by measuring the induction of nitric oxide production. One and 2-day thymulin treatment at 10 and 100ng per 100g body weight doses significantly increased responsiveness to ChIFN-gamma while 1ng per 100g body weight had no effect. Other experiments compared the effect of thymulin treatments in Cornell K strain chickens, having normal serum thymulin levels with sex-linked dwarf (SLD) chickens which are deficient in serum thymulin. The dose of thymulin treatment required to significantly increase responsiveness to ChIFN-gamma differed between strains. Finally, the effect of direct in vitro thymulin treatments on macrophage responsiveness to ChIFN-gamma was evaluated. There were no significant increases in responsiveness to ChIFN-gamma between treatment groups within the macrophage cell line, HD-11, when cultured in the presence of 0-200pg thymulin/ml. These data suggest that the effect of thymulin on AEC responsiveness to ChIFN-gamma is indirectly mediated.

Animals↗
PubMed2002

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system.

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.

Animals↗
PubMed2001

Conservation of coding potential and terminal sequences in four different isolates of Borna disease virus.

We determined the complete nucleotide sequences of two poorly characterized strains of Borna disease virus (BDV) and compared them to reference strains V and He/80. Strain H1766 was almost 98% and 95% identical to strains V and He/80, respectively, whereas strain No/98 was only about 81% identical to both reference strains. In contrast to earlier reports, we found an additional A residue at the extreme 3'-end of the single-stranded RNA genome in all four BDV strains. The exact numbers of nucleotides in the four BDV genomes could not be determined due to a micro-heterogeneity at the 5'-end. If our longest sequence is a correct copy of the viral RNA, the two ends of the BDV genome would show almost perfect complementarity. All three transcription start sites, all four termination sites, both splice donor sites and both major splice acceptor sites are highly conserved, whereas a minor alternative splice acceptor site is not. The L protein of No/98 differs at 7% of its amino acid positions from the polymerase in the other strains, with most differences mapping to the C-terminal moiety of the molecule. Re-evaluation of L protein sequences of strains V and He/80 revealed differences at several positions compared to published information, indicating that variant forms of the viral polymerase have previously been characterized. These results are important because correct structures of genome ends and of the polymerase gene are the most critical parameters for the future development of techniques that will permit the genetic manipulation of BDV.

3' Untranslated Regions↗
PubMed2000

Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. It uses the cellular splicing machinery to generate a set of alternatively spliced mRNAs from the 2.8 and 7.1 kb primary transcripts, each harbouring two introns. To determine whether splicing of these transcripts is regulated by viral factors, the extent of splicing was studied in infected cells and COS-7 cells transiently transfected with plasmids encoding the 2.8 kb RNA of BDV. Unspliced RNA was found to be the most abundant RNA species in infected cells, whereas viral transcripts lacking both introns were only found in minute amounts. In sharp contrast, plasmid-derived 2.8 kb RNA was predominantly intron 1-spliced and double-spliced. Co-expression of the BDV proteins P, N and X did not influence splicing of plasmid-expressed 2.8 kb RNA. Furthermore, the splicing pattern did not change when the 2.8 kb RNA was expressed in BDV-infected cells. Based on these results we speculate that splicing of authentic BDV transcripts is tightly linked to transcription by the viral polymerase.

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