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.

Hintergrund: Neben verschiedenen Organmanifestationen weist Borrelia burgdorferi eine hohe Neurotropie mit unterschiedlichen Manifestationen wie Meningitiden bzw. Meningoencephalitiden, spinale Myelitiden, in Einzelfällen auch cerebrale Vasculitiden oder kraniale Hämorrhagien mit multifokalen oder periventrikulären cerebralen Läsionen auf. Über die pathophysiologischen Hintergründe besteht Uneinigkeit.

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.

The La protein was recently identified as a host factor potentially involved in the cytokine-induced post-transcriptional down-regulation of hepatitis B virus (HBV) RNA. The La binding site was mapped to a predicted stem-loop structure within a region shared by all HBV RNAs, and it was concluded that the La protein might be an HBV RNA-stabilizing factor. To characterize the RNA binding mediated by the different RNA recognition motifs (RRMs) of the human La protein, several La deletion mutants were produced and analyzed for HBV RNA binding ability. The data demonstrate that the first RRM is not required for binding, whereas the RNP-1 and RNP-2 consensus sequences of the RRM-2 and RRM-3 are separately required for binding, indicating a cooperative function of these two RRMs. Furthermore, the results suggest that multimeric La disassembles into monomeric La upon binding of HBV RNA.B. By gel retardation assay the affinity of the wild type human La.HBV RNA.B interaction was determined in the nanomolar range, comparable to the affinity determined for the mouse La.HBV RNA.B interaction. This study identified small regions within the human La protein mediating the binding of HBV RNA. Hence, these binding sites might represent targets for novel antiviral strategies based on the disruption of the human La.HBV RNA interaction, thereby leading to HBV RNA degradation.

It is well established that interferon-alpha can induce non-cytotoxic intracellular suppression of hepatitis B virus replication, but the mechanisms involved are unclear. Cell culture studies to characterize these mechanisms are restricted, in part because hepatitis B virus replicates almost exclusively in liver-derived cells. To overcome this limitation we used a cytomegalovirus promoter-controlled hepatitis B virus expression system, which leads to intracellular viral replication even in non-hepatic cell lines. In this experimental system interferon-alpha treatment specifically suppressed viral replication demonstrating that antiviral activities against hepatitis B virus are not restricted to hepatic cells. Furthermore, the interferon-inducible MxA protein was recently reported to play a key role in the antiviral action of interferon-alphaa against hepatitis B virus. Our data demonstrate that interferon-alpha also suppresses hepatitis B virus replication in MxA-deficient HEp2 cells, indicating that MxA is not essential for these activities. Taken together, our data imply that the experimental approach presented can also be adapted to established cell lines which are deficient in parts of the signal transduction pathway or other elements located further downstream, providing important insights into mechanisms specifically suppressing hepatitis B virus.

ABSTRACT 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.

The antiviral effect of interferon-alpha (IFN alpha) 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-3 gamma (p48) has been identified in the HBV enhancer I region. Mutation of this element was shown to abrogate IFN alpha -mediated reduction of HBV X-gene promoter-driven reporter gene expression. This suggested a role of the ISRE and of p48 in IFN alpha -induced antiviral activity against productive HBV infection. Here, we analyzed the antiviral effect of both IFNa and enhanced p48 expression on complete HBV genomes containing the wildtype or mutated ISRE. In human hepatoma cells transfected with both genomes, viral RNA and replicative intermediates were reduced by IFNa treatment to a similar degree. Enhanced p48 expression increased IFN alpha -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 IFNa 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,

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.

Background/Aims: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN alpha), which results in efficient reduction of the viral load only in 10-20% of treated patients. The mechanisms induced by IFN alpha resulting in reduction of viremia in responding patients are unknown. The aim of this study was to characterize HBV-specific IFN alpha-induced intracellular inhibitory mechanisms and IFN alpha-sensitive HBV targets.Methods: To determine the antiviral activity, cells transiently transfected with HBV DNA were treated with IFNa 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 IFN alpha-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 tar-get region mediating IFN alpha-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 IFN alpha-induced antiviral mechanisms and HBV targets. At least two IFN alpha-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 IFN alpha-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.