Identifizierung einer O-Glykosylierungsstelle der Woodchuck Hepatitis Virus (WHV) preS2-Domäne und deren Einfluß auf den intrazellulären Transport des middle surface antigen (WHmsAg)

Im Rahmen dieser Arbeit sollte die Glykosylierung des middle surface antigen des Woodchuck Hepatitis Virus untersucht werden. Dazu wurden chimäre Proteine aus der WHVpreS2/S-Region und dem S-Antigen von HBV mit der HBV ´a´-Determinante konstruiert. In der preS2-Region konnte durch zielgerichtete Mutagenese und in vitro Translations-Assays eine O-Glykosylierungsstelle an Position 5 (Thr) der Aminosäuresequenz identifiziert werden. Mit Hilfe von Immunfluoreszenzfärbungen und Konfokalmikroskopie wurde gezeigt, daß die Glykosylierung des preS2-Proteins eine wichtige Rolle für den intrazellulären Transport dieses Proteins spielt. Dabei wurde deutlich, daß falsche oder fehlende Glykosylierung zu einer ringförmigen Anreicherung des preS2-Proteins führt. Diese Ringbildung erfolgt nach den vorliegenden Daten zwischen dem endoplasmatischen Retikulum und dem Golgi-Apparat, also in dem zellulären Kompartiment, das für die O-Glykosylierung verantwortlich ist. Dies ist eine mögliche Erklärung für den Erfolg einer Therapie mit Glykosylierungsinhibitoren. Vergleichende Studien mit einer preS2-minus Chimäre zeigten, daß die Ringbildung nur bei fehlender Glykosylierung des preS2-Proteins auftritt, nicht aber bei nicht-Translation der preS2-Domäne. Der Therapieerfolg mit Glykosylierungsinhibitoren im Falle einer Infektion mit HBV-Varianten, denen das preS2-Protein fehlt, ist also fraglich. Desweiteren sollte die Replikationskompetenz von WHV-Stämmen mit Glykosylierungsdefekt sollte mit Hilfe in vivo Transfektionen untersucht werden. Im Rahmen dieser Versuche konnte jedoch allenfalls die kurzfristige Expression viraler Proteine nachgewiesen werden, nicht aber die Replikation der glykosylierungsdefekten oder preS2minus Mutante. Ein Virustiter und damit Replikation konnte nicht nachgewiesen werden, und zwar weder für die preS2minus-Mutante, noch für die Variante mit dem Glykosylierungsdefekt. Im zweiten Teil der Arbeit wurde eine RNase Protection Assay etabliert. Mit Woodchuck-spezifischen Sonden, sogenannten Riboprobes, kann die Expression der Cytokine IFN-g, TNF-a und IL-15 sowie der T-Zellmarker CD3, CD4 und CD8 qualitativ und quantitativ untersucht werden. Es wurde gezeigt, daß die Marker CD3, CD4 und CD8 während der chronischen Infektion stärker exprimiert werden als in der Akutphase der Infektion. Alle untersuchten Cytokine und T-Zellmarker sind während der chronischen Infektion natürlichen Schwankungen unterworfen. Ausgehend von einem Mittelwert betragen diese Schwankungen ± 50%, gemessen in DLU. Diese Fluktuationen sind zehnmal so hoch wie die Schwankungen, die man beobachtet, wenn man zu einem einzigen Zeitpunkt multiple Biopsien aus verschiedenen Regionen einer einzigen Leber untersucht. Ebenfalls konnten unterschiedliche Expressionsmuster bei verschiedenen Tiere beobachtet werden. Der RNase Protection Assay konnte bereits in mehreren Projekten erfolgreich zur Untersuchung der Cytokin- und T-Zellmarker-Expression eingesetzt werden. So konnte gezeigt werden, daß das Immunmodulans a-Galaktosyl-Ceramid Spezies-spezifisch wirkt, und im Woodchuck nicht zu einer Erhöhung der Interferon-g Expression führt. Mit Hilfe des RPA konnte darüber hinaus die Erhöhung der CD4, CD8 und Interferon-g Expression nach einer Tumortherapie mit einem rekombinanten Adenovirusvektor nachgewiesen werden. Die Erhöhung der Expression dieser Marker erfolgte durch einen Gentransfer der Gene für IL-12 und B7.1. Darüber hinaus sind seit kurzem Interferon-a und GAPDH aus dem Woodchuck als Konstrukte für den RNase Protection Assay verfügbar (Daten nicht gezeigt, Klonierung erfolgte analog zu 3.14), wodurch weitere Untersuchungen der Woodchuck-Immunantwort ermöglicht werden. Durch die Expression des Interferon-g-Rezeptors des Murmeltiers wurde der Grundstein für weitere Untersuchungen der Interferon-g Wirkung gelegt. Aufgrund der bisherigen Daten müßte es daher in absehbarer Zukunft möglich sein, weitere immunologisch-virologische Studien der hepadnaviralen Infektion im natürlichen Infektionsmodell durchzuführen

Latent infection of human bocavirus accompanied by flare of chronic cough, fatigue and episodes of viral replication in an immunocompetent adult patient, cologne, Germany

Publisher Copyright: © 2016 The Authors.Introduction: The human bocavirus (HBoV) is a parvovirus and is associated with mild to lifethreatening acute or persisting respiratory infections, frequently accompanied by further pathogens. So far, there is limited knowledge on the mechanisms of persistence, and no reports on chronic infections or latency have been published so far. Case presentation: An immunocompetent male patient suffers from a chronic HBoV1 infection, i.e. viral DNA was detected in both serum and bronchoalveolar lavage (BAL) for >5 months without co-infections and with respiratory symptoms resolved spontaneously while receiving symptomatic treatment with montelukast and corticosteroids. Following the symptomatic medication of a chronic infection with HBoV1 viraemia indicating active viral replication lasting over 5 months, the patient cleared the viraemia and no further viral DNA was detectable in the BAL. However, by fluorescence in situ hybridization analyses of mucosal biopsies, it was shown that the virus genome still persisted in the absence of viral shedding but in a more compact manner possibly representing a supercoiled episomal form of this otherwise linear singlestranded DNA genome. This indicated the entry into a latency phase. Moreover, the cytokine profile and the IP-10/TARC ratio, a marker for fibrotization, seem to have been altered by HBoV1 replication. Although specific IgG antibodies were detectable during the whole observation period, they showed an apparently insufficient neutralising activity. Conclusion: On the one hand, these findings suggest that the symptomatic medication may have led to clearance of the virus from blood and airways and, moreover, that the viral DNA persists in the tissue as an altered episomal form favoured by lacking neutralising antibodies. This appears to be important in order to reduce possible long-term effects such as lung fibrosis.publishersversionPeer reviewe

Utility of two novel multiplexing assays for the detection of gastrointestinal pathogens – a first experience

BACKGROUND: Cause for gastroenteritis range from viral, bacterial to parasitic pathogens. Rapid Multiplexing techniques like ProGastro_SSCS and xTAG_GPP can detect broad panels of pathogens simultaneously. We performed a field test with a total number of 347 stool samples from adult hospitalized patients that were tested with the Luminex xTAG GPP assay; of the 157 samples positively tested for at least one pathogen by xTAG GPP a total number of 30 samples was retested with the ProGastro SSCS assay. Assays were compared to standard routine diagnostics. FINDINGS: Multiplexing significantly reduced the time to the initial identification of a pathogen. Moreover, multiplexing detected pathogens for which a diagnostic assays was not requested by the physician and thus may be an important tool for avoiding nosocomial outbreaks. CONCLUSION: This first frontline approach with these assays approves their utility compared to conventional microbiological methods

Novel mutation in YMDD motif and direct neighbourhood in a child with chronic HBV-infection and clinical lamivudine and adefovir resistance - a scholarly case

<p>Abstract</p> <p>Context</p> <p>Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) which meanwhile has become the 5^thmost reason for a fatal outcome of cancer. Worldwide, approximately 350 million people are chronically HBV infected and as such of risk to develop HCC, of those an estimated high rate of children. Treatment of chronic infection is sufficient to reduce the rate of HCC but the rate of sustained virological response remains to low, not at least due to emergence of resistant virus strains. Less is known on HBV infection in children despite the extremely high rate of chronicity.</p> <p>Objective, Design, Setting, and Patient</p> <p>The case of a nine years old male with a 6 year history of chronic HBV infection, of those 5 years with antiviral treatment is described.</p> <p>Interventions and Main Outcome Measure(s)</p> <p>Before our lab was consulted, the patient was unsuccessfully treated with interferon, an obscure drug named Hepon, which should activate antiviral immune response, and Lamivudine, the latter most likely becoming ineffective due to the mergence of resistant subpopulations (rtL180 M, rtV207 M, two strains with stop codons at position rt188 and rt198, rtM204V (YVDD), rtM204K (YKDD)). Replacement of Lamivudine by adefovir displayed no advantage despite the lack of resistance mutations, thus no decrease in viremia was observed under adefovir treatment.</p> <p>Results and Conclusions</p> <p>Novel mutations in the YMDD motif and its direct neighbourhood were observed, both being compatible with Lamivudine resistance. No mutations were found that are associated with ADF resistance. Both, the clinical course of treatment and the genotypic resistance profile emphasize the need for systematic analyses of the HBV resistance mechanisms and structured therapy concept also for children chronically infected with HBV.</p

Does human bocavirus infection depend on helper viruses? A challenging case report

A case of severe diarrhoea associated with synergistic human bocavirus type 1 (HBoV) and human herpes virus type 6 (HHV6) is reported. The case supports the hypotheses that HBoV infection under clinical conditions may depend on helper viruses, or that HBoV replicates by a mechanism that is atypical for parvoviruses, or that HBoV infection can be specifically treated with cidofovir

Selection and counterselection of the rtI233V adefovir resistance mutation during antiviral therapy

Recently, we reported on three patients with chronic hepatitis B virus (HBV) infection for whom adefovir (ADF) therapy virologically failed, most likely due to a preexisting rtI233V HBV polymerase mutation. Here, we describe two further patients with chronic HBV infection who were found to develop the rtI233V mutation after initiation of ADF therapy. These patients represent the first cases known so far in which the rtI233V ADF resistance mutation evolved under persistent HBV replication during HBV therapy with ADF. Interestingly, one of the previously described patients, who was initially successfully switched from ADF to tenofovir (TDF) and became virologically suppressed subsequently, experienced a moderate but remarkable rebound of HBV viremia after switching from TDF to entecavir, due to the emergence of renal toxicity. Thus, we provide evidence for the selection and counterselection of the rtI233V ADF resistance mutation during antiviral therapy

Human Bocavirus – Insights into a Newly Identified Respiratory Virus

Human Bocavirus (HBoV) was discovered in 2005 using a molecular virus screening technique. It is often found in respiratory samples and is a likely cause for respiratory diseases in children. HBoV is distributed worldwide and has been found not only in respiratory samples, but also in feces, urine and serum. HBoV infections are mostly found in young children and coinfections with other respiratory viruses are often found, exacerbating the efforts to link HBoV to specific symptoms. The purpose of this review is to give an overview of recent HBoV research, highlighting some recent findings

Is there Emergence of Clinical HBV Resistance Under Long-Term HBV Combination Therapy? A Challenging Case Report

A first case of clinical tenofovir (TDF) HBV resistance in an HIV/HBV coinfected patient who developed an acute flare of hepatitis B is reported. The clinical course was accompanied by signs of acute liver failure after being on successful HBV treatment with tenofovir and persistently undetectable HBV-DNA viral load for over five years