Non-human primates in outdoor enclosures: risk for infection with rodent-borne hantaviruses.

Different species of non-human primates have been exploited as animal disease models for human hantavirus infections. To study the potential risk of natural hantavirus infection of non-human primates, we investigated serum samples from non-human primates of three species living in outdoor enclosures of the German Primate Center (GPC), Göttingen, located in a hantavirus endemic region of central Germany. For that purpose we used serological assays based on recombinant antigens of the bank vole (Myodes glareolus) transmitted Puumala virus (PUUV) and the common and field vole (Microtus arvalis, Microtus agrestis) associated Tula virus (TULV) which are both broadly geographically distributed in Germany. In 24 out of 251 (9.6%) monkey sera collected in 2006 PUUV- and/or TULV-reactive immunoglobulin G (IgG) antibodies were detected. Investigation of follow-up sera from 13 animals confirmed for two animals a seroconversion due to hantavirus exposure at the GPC. To prove the origin of the infection, wild rodents from the surrounding regions were analyzed by hantavirus-specific reverse transcriptase-PCR analysis. In 6 of the 73 investigated bank voles and 3 of the 19 investigated Microtus spp. PUUV- and TULV-specific nucleic acid sequences, respectively, were detected. In conclusion, our investigations demonstrate for the first time natural infections of non-human primates in outdoor enclosures in Germany. These findings highlight the importance of hantavirus surveillance in those primate housings and corresponding preventive measures against wild rodents, particularly in hantavirus endemic regions.

Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame.

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

Lack of a role of the interferon-stimulated response element-like region in interferon alpha -induced suppression of Hepatitis B virus in vitro.

The antiviral effect of interferon-alpha (IFNalpha) on hepatitis B virus (HBV) is well documented in vitro and in vivo, but the mechanisms involved are elusive. Recently, an interferon-stimulated response like element (ISRE) competent for binding of interferon-stimulated gene factor-3gamma (p48) has been identified in the HBV enhancer I region. Mutation of this element was shown to abrogate IFNalpha-mediated reduction of HBV X-gene promoter-driven reporter gene expression. This suggested a role of the ISRE and of p48 in IFNalpha-induced antiviral activity against productive HBV infection. Here, we analyzed the antiviral effect of both IFNalpha and enhanced p48 expression on complete HBV genomes containing the wild-type or mutated ISRE. In human hepatoma cells transfected with both genomes, viral RNA and replicative intermediates were reduced by IFNalpha treatment to a similar degree. Enhanced p48 expression increased IFNalpha-induced suppression of HBV RNA significantly from 75 +/- 22.5% to 46 +/- 9.8%, but this was independent of the integrity of the ISRE-like region. These data imply that p48 neither mediates the antiviral activity of IFNalpha against HBV nor down-regulates enhancer I activity by binding directly to the HBV ISRE-like region, but rather argue for an indirect role of p48.

The tetracycline-responsive promoter contains functional interferon-inducible response elements.

Tetracycline (tet)-responsive expression vectors allow controlled inducible expression of proteins in mammalian cells. This system is widely used for experimental research both in vivo and in vitro. In our attempts to use this system to study the antiviral effect of IFNalpha on hepatitis B virus, we discovered an unexpected feature of the tet-responsive promoter (tet promoter) of the currently available expression vectors. IFNalphawas found to stimulate tet promoter activity after transient transfection in a dose- and cell type-dependent manner. By sequence inspection, an IFNalpha-stimulated response element (ISRE)-like sequence was identified in the linker regions located between the heptameric tet operator sequences. Gel shift assays revealed binding of IFN-stimulated gene factors to these sequences, indicating that they mediate the IFNalpha-mediated promoter stimulation. These data demonstrate an unexpected feature of the tet-responsive expression system which needs to be taken into account when using this system for analysis of cytokine functions in vitro and in vivo. The data also imply that the tet promoter-based expression system can be rendered non-responsive to IFNalpha by mutagenesis of the ISREs and this may be essential when considering gene therapy in vivo.

Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFNalpha), which results in efficient reduction of the viral load only in 10-20% of treated patients. The mechanisms induced by IFNalpha resulting in reduction of viremia in responding patients are unknown. The aim of this study was to characterize HBV-specific IFNalpha-induced intracellular inhibitory mechanisms and IFNalpha-sensitive HBV targets. METHODS: To determine the antiviral activity, cells transiently transfected with HBV DNA were treated with IFNalpha and thereafter, viral products were quantified at different time points. RESULTS: Time-dependent reduction of RNA, replicative DNA-intermediates, core protein and secreted HBsAg/HBeAg levels was observed in IFNalpha-treated cells. Viral RNA levels were reduced most effectively early post-treatment whereas those of core protein and replicative intermediates decreased later. By expression of subgenomic HBV sequences, an RNA target region mediating IFNalpha-induced RNA degradation was mapped. CONCLUSIONS: These data indicate that HuH7 cells transiently transfected with HBV-DNA represent a system well suited for detailed analysis of IFNa-induced antiviral mechanisms and HBV targets. At least two IFNalpha-induced HBV-specific antiviral activities are active in this system: one reduces the levels of core protein and replicative intermediates, the other leads to posttranscriptional degradation of HBV-RNA. Based on the established in vitro system a detailed characterization of the IFNalpha-sensitive RNA-region and of factors mediating this intracellular antiviral effect is feasible. This may lead to the development of novel strategies for therapy of chronic hepatitis.

Interferon-gamma receptor-mediated but not tumor necrosis factor receptor type 1- or type 2-mediated signaling is crucial for the activation of cerebral blood vessel endothelial cells and microglia in murine Toxoplasma encephalitis.

The regulatory role of interferon-gamma receptor (IFN-gammaR)- and tumor necrosis factor receptor (TNFR)-mediated immune reactions for the activation of cerebral endothelial cells, microglia, and astrocytes was evaluated in a model of murine Toxoplasma encephalitis (TE). Brain endothelial cells of wild-type mice reacted in response to Toxoplasma infection with a strong up-regulation of the vascular cell adhesion molecule, the intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I and II antigens. A similar response was seen in mice genetically deficient for either TNFR1, TNFR2, or both TNFRs, whereas IFN-gammaR-deficient (IFN-gammaR0/0) mice were found to be defective in the up-regulation of these molecules. However, recruitment of leukocytes to the brain and their intracerebral movement were not impaired in IFN-gammaR0/0 mice. In addition, microglia of Toxoplasma gondii-infected IFN-gammaR0/0 mice failed to induce expression of ICAM-1, leukocyte function-associated antigen (LFA)-1, and MHC class I and II antigens, whereas wild-type and TNFR-deficient mice up-regulated these molecules. Moreover, TNF-alpha mRNA production of F4/80(+) microglia/macrophages was impaired in IFN-gammaR0/0 mice, but not in TNFR-deficient mutants. However, induction of interleukin (IL)-1beta, IL-10, IL-12p40, and IL-15 mRNA was independent of IFN-gammaR and TNFR signaling. In conclusion, IFN-gammaR, but not TNFR signaling, is the major pathway for the activation of endothelial cells and microglia in murine TE. These findings differ from observations in other inflammatory central nervous system disorders, indicating specific regulatory mechanisms in this parasitic cerebral infection.

Apoptosis and apoptosis-related gene products in primary non-Hodgkin's lymphoma of the central nervous system.

The incidence of primary lymphomas of the central nervous system (CNS) has significantly increased over the last years. However, the pathogenesis of this serious and fatal disease is still largely unknown. The aim of the present study was to investigate whether impairment of apoptosis is involved in the pathogenesis of primary CNS lymphomas. A series of 35 primary CNS lymphomas was investigated for the presence of apoptotic cells and the expression of apoptosis-inhibiting and proapoptotic gene products of the bcl family by application of the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) technique and immunohistochemistry. The majority (23/35) of the tumors contained no or less than 10% of apoptotic cells. All tumors were MIB-1 positive, and 53% of them showed a high proliferative activity with more than 20% MIB-1-positive cells. The bcl-2 gene was expressed in 54% of the tumors (19/35), whereas bcl-x and bax gene products were present in only a low fraction of these lymphomas (4/35). In contrast, bak and the tumor suppressor gene p53 product were not detectable. These findings indicate that apoptosis is inhibited in the majority of this series of primary CNS lymphomas. Since there was no statistical correlation between the degree of apoptosis and the expression of proteins of the bcl gene family, other apoptosis-inhibiting factors may be involved in the pathogenesis of primary CNS lymphomas.

Crucial role of TNF receptor type 1 (p55), but not of TNF receptor type 2 (p75), in murine toxoplasmosis.

TNF-alpha exerts its biologic activity through two distinct receptors, TNF receptor type 1 (TNFR1, p55) and TNF receptor type 2 (TNFR2, p75). To analyze their function in toxoplasmosis, we orally infected mice genetically deficient for TNFR1 (TNFR1(0/0)), TNFR2 (TNFR2(0/0)), or both TNF receptors (TNFR1/2(0/0)), as well as wild-type (wt) mice with a low-virulent strain of Toxoplasma gondii. TNFR1/2(0/0) and TNFR1(0/0) mice succumbed to toxoplasmosis within 17 and 27 days, respectively, whereas TNFR2(0/0) and wt mice were equally resistant to acute toxoplasmosis. Histopathology attributed death of TNFR1/2(0/0) and TNFR1(0/0) mice to a fulminant necrotizing encephalitis. In addition, pneumonia contributed to the fatal outcome. The poor prognosis of TNFR1/2(0/0) and TNFR1(0/0) mice was reflected by a significantly increased parasitic load in the brain and lung as compared with TNFR2(0/0) and wt mice. Immunohistochemistry demonstrated a remarkable reduction of inducible nitric oxide synthase protein in brain and lung of TNFR1/2(0/0) and TNFR1(0/0) as compared with TNFR2(0/0) and wt mice. Reverse-transcribed PCR showed that in contrast to TNFR2(0/0) and wt mice, TNFR1(0/0) mice were unable to up-regulate inducible nitric oxide synthase mRNA transcripts in the course of infection, whereas intracerebral levels of IFN-gamma, TNF-alpha, and IL-1beta mRNA transcripts, recruitment of immune cells to the brain, and the amount of apoptotic cells in inflammatory foci did not differ significantly among the various experimental groups. These results illustrate that in Toxoplasma encephalitis, TNF-alpha-mediated immune responses are of crucial importance and that signaling through TNFR1, but not TNFR2, provides the stimulus required for the induction of protective nitric oxide.

Interleukin-10 downregulates the intracerebral immune response in chronic Toxoplasma encephalitis.

The expression of the immunosuppressive cytokine interleukin (IL)-10 in the normal and Toxoplasma gondii-infected murine brain was analysed. Microglia/macrophages expressed IL-10 at the mRNA and protein level in the normal brain. In Toxoplasma encephalitis (TE), CD4+ and CD8+ T-cells also contributed to the upregulated IL-10 production. Neutralization of endogenous IL-10 in chronic TE reduced the intracerebral parasitic load and increased the number of immune cells and the production of protective cytokines. These findings indicate that intracerebral expression of IL-10 interferes with the immune response in TE and may contribute to parasite persistence in the brain.

Interferon-gamma receptor-deficiency renders mice highly susceptible to toxoplasmosis by decreased macrophage activation.

Toxoplasma gondii may cause severe infections in immunocompromised patients including fetuses and those with AIDS. Among the factors mediating protection against T. gondii, IFN-gamma has gained special attention. To analyze the role of IFN-gamma in the early phase of toxoplasmosis, IFN-gamma receptor-deficient (IFN-gamma R0/0) mice were orally infected with low-virulent toxoplasms. IFN-gamma R0/0 mice died of the disease up to day 10 postinfection, whereas immunocompetent wild-type (WT) mice developed a chronic toxoplasmosis. Histopathology revealed that in IFN-gamma R0/0 mice, the parasite multiplied unrestrictedly in the small intestine, the intestinal lymphatic tissue, the liver, and the spleen. Ultimately, animals died of a necrotizing hepatitis. In WT mice, the same organs were effected, but multiplication of the parasite was effectively limited. Compared with WT mice, immunohistochemistry and flow cytometry demonstrated that in IFN-gamma R0/0 mice, macrophages were only marginally activated in response to the infection, as evidenced by a reduced expression of major histocompatability complex class II antigens. In addition, immunohistochemistry and RT-PCR showed a reduced production of the macrophage-derived cytokines tumor necrosis factor-alpha, inducible nitric oxide synthase, and IL-1 beta in the liver of IFN-gamma R0/0 mice. In contrast, activation of T cells, recruitment of immune cells to inflammatory foci, and anti-T. gondii IgM antibody production were unaffected by the mutation of the IFN-gamma R. Moreover, induction of IL-2, IL-4, and IL-10 mRNA transcripts in the liver was normal in IFN-gamma R0/0 mice. Adoptive transfer experiments revealed that the immune T cells of WT animals did not protect IFN-gamma R0/0 mice from lethal infection with highly virulent toxoplasms, whereas WT mice were significantly protected by the adoptive transfer. Based on these studies, we conclude that IFN-gamma is absolutely required for an efficient activation of macrophages. Macrophages are of critical importance in toxoplasmosis, and insufficient macrophage activation cannot be compensated by other immune mechanisms.