Investigations into the Integration of Nucleotide Sequences of the Reticuloendotheliosis Provirus into the Genome of Fowlpox Virus

Deckblatt-Impressum Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung und Ziele der Arbeit Literaturübersicht Material und Methoden Versuchsdurchfürung und Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Danksagung Lebenslauf SelbständigkeitserklärungErstes Ziel der vorliegenden Arbeit waren umfangreiche molekularbiologische Untersuchungen an Hühnerpocken (FPV) DNAs aus Feldausbrüchen der letzten Jahre, um festzustellen, in welchem Umfang REV Nukleotidsequenzen in das Genom integriert vorlagen. Dazu wurde eine Multiplex PCR zum gleichzeitigen Nachweis des 4b Core Proteins des FPV, der gag , pol und env Gene des REV sowie der long terminal repeats (LTR) des Retikuloendotheliose Virus (REV) etabliert. In fast allen untersuchten Feld DNAs konnten alle vier REV spezifischen Sequenzbereiche und zusätzlich ein chimäres FPV REV PCR Produkt nachgewiesen werden, was für ein Vorliegen eines in das FPV Genom integrierten fast vollständigen REV Provirus (fvRP) spricht. Mittels Long Distance PCR wurde bei drei Feld DNAs versucht, dies zu bestätigen. Der Nachweis war mit FPV spezifischen Primern, die die Integrationsstelle des fvRP umfassen, nicht möglich, sondern nur mit REV LTR spezifischen Primern. Durch die sich an die Long Distance PCR von LTR zu LTR anschließende Restriktionsenzymanalyse wurde gezeigt, daß das Amplikon dem REV Provirus entsprach. Ferner wurden die Integrationsstelle des fvRP einer Feld DNA und des Vakzinestammes HP B sowie das FPV und REV Sequenzen umfassende chimäre PCR Produkt der Feld DNA sequenziert. So konnte auch in dem Vakzinestamm ein ca. 300 bp großer Überrest der LTR nachgewiesen werden. Der Vergleich aller drei erhaltenen Sequenzen mit entsprechenden veröffentlichten Sequenzen zeigte lediglich eine Punktmutation. Insgesamt konnte eine hohe Konserviertheit der Sequenzen bei Stämmen aus drei Kontinenten festgestellt werden. Zur Bestimmung des Verhältnisses zwischen FPV spezifischer DNA und REV provirusspezifischer DNA in einigen Feld DNAs und in verschiedenen in vitro Passagen eines Feldisolates wurde eine qPCR etabliert. Bei allen untersuchten Feld DNAs lag die Subpopulation von FPV Virionen mit integriertem fvRP in großer Überzahl vor. Die Untersuchung verschiedener in vitro Passagen zeigte einen annähernd exponentiellen Verlust des fvRP mit einer Verlustrate von ca. 0,5 (50 %) zwischen dem Originalmaterial und der 16. Passage. Nach 32 Passagen war mittels PCR keine REV spezifische DNA mehr nachweisbar. Des weiteren wurden ausführliche serologische Untersuchungen von Feldseren auf das Vorkommen von Antikörpern gegen FPV sowie REV durchgeführt. Da kein kommerzieller ELISA zum Nachweis von Antikörpern gegen FPV zur Verfügung stand, wurde ein indirekter ELISA entwickelt. Die Sensitivität betrug 85,3 %, die Spezifität 100 %. Anschließend wurde die Wiederholbarkeit der im ELISA erzielten Ergebnisse untersucht. Der Intraassay Vergleich zeigte eine gute Wiederholbarkeit. Dagegen brachte der Interassay Vergleich unbefriedigende Ergebnisse. Beim Vergleich mit anderen Methoden zum Nachweis von Antikörpern gegen FPV erwies sich der indirekte Immunfluoreszenztest als geringfügig sensitiver als der ELISA. Der AGP stellte sich als weniger sensitiv heraus. Die Untersuchung von Seren aus an FPV erkrankten Herden auf Antikörper gegen FPV und REV zeigte bei etwa der Hälfte der Tiere Antikörper gegen FPV und bei einem größeren Anteil der Tiere Antikörper gegen REV, wobei zwischen den Herden z. T. deutliche Unterschiede auftraten. Ein vergleichbarer Anteil von Seren aus gegen FPV geimpften Herden hatte Antikörper gegen FPV, aber nur in zwei der zehn untersuchten Herden hatten Tiere Antikörper gegen REV. Dies macht deutlich, daß die Bildung von REV Antikörpern von dem integrierten fvRP und nicht durch eine zufällige, gleichzeitige Infektion mit REV induziert wurden, und daß die Fähigkeit, Antikörper gegen REV zu induzieren, bei deutschen FPV Feldstämmen weit verbreitet ist. In zwei Infektionsversuchen wurde die Antikörperbildung gegen FPV und REV nach Infektion mit unterschiedlichen Zellkulturpassagen eines FPV Feldisolats und dem Vakzinestamm HP B untersucht. In beiden Versuchen induzierte das Feldisolat in einer niedrigen Passage, in der das fvRP noch mittels PCR nachweisbar war, nach intrakutaner Infektion keine Antikörper gegen FPV, aber, abhängig von der Infektionsdosis, bei einem unterschiedlich hohen Anteil der Tiere Antikörper gegen REV. Dagegen induzierte der Vakzinestamm, unabhängig von der Infektionsdosis, bei fast allen Tieren Antikörper gegen FPV, aber keine gegen REV. Das Feldisolat in einer hohen Passage, in der das fvRP mittels PCR nicht mehr nachweisbar war, induzierte bei einem geringen Anteil der Tiere Antikörper gegen FPV und keine gegen REV. Deswegen wurde vermutet, daß das integrierte fvRP zur Hemmung der Antikörperbildung gegen FPV nach einer Infektion mit einem Feldisolat beiträgt. Im ersten Versuch wurden die Tiere nach fünf Wochen intravenös mit der niedrigen Passage des Feldisolates reinfiziert. Daraufhin entwickelte etwa die Hälfte der Tiere aus den Gruppen, die zunächst auch das Feldisolat erhalten hatten, Antikörper gegen FPV. Der Anteil der Tiere mit Antikörpern gegen REV erhöhte sich geringgradig. Bei den Tieren, die zuvor vakziniert worden waren, war keine Antikörperreaktion gegen REV feststellbar. Dies kann darauf zurückzuführen sein, daß durch die vorangegangene Immunisierung mit dem Vakzinestamm eine Replikation des FPV Feldstammes und somit eine Bildung von Antikörpern gegen REV verhindert wurden. Nach der intrakutanen Infektion mit der niedrigen Passage des Feldisolates und nach der intravenösen Reinfektion ließ sich in den peripheren Blut mononukleären Zellen REV spezifische DNA, aber bis auf eine Ausnahme, keine FPV spezifische DNA nachweisen. Dies deutet auf eine Bildung infektiösen REVs aus dem FPV Feldisolat mit dem integrierten fvRP hin.The first aim of this study was to find out by molecularbiological methods to which extent REV specific DNA was integrated into the DNA of fowlpox virus (FPV) that caused field outbreaks in Germany during the recent years. For this purpose a multiplex PCR, which detects the gene of the 4b core protein of FPV as well as gag , pol und env genes of REV and the long terminal repeats of REV, was established. In most investigated DNAs the four sequences specific for REV were detected. Additionally a chimeric FPV REV PCR product was found in most DNAs. This indicates the presence of an integrated, almost complete REV provirus (fvRP). In addition three DNAs were investigated further by long distance PCR to confirm this. The detection of the integrated fvRP was not possible with primers specific for FPV that flank the integration site of the fvRP, but only with primers specific for the LTR of REV. By adjacent restriction enzyme analysis the result was confirmed. Furthermore the integration site of the fvRP in a field DNA and in the vaccine strain HP B as well as the chimeric FPV REV PCR product from the field DNA were sequenced. By these means a remnant of the LTR of about 300 bp was detected also in the vaccine strain. Comparison of all three obtained sequences with published sequences showed only one single base pair substitution. This demonstrated a high conservation of the integration site of FPV strains from three continents. A qPCR was established to determine the ratio between DNA specific for FPV and DNA specific for REV in some field DNAs and in different passages in vitro of a field isolate. In all investigated field DNAs the FPV subpopulation with integrated fvRP predominated. When passaged in vitro, the field isolate lost the fvRP at a rate of about 0.5 (50 %) until the 16th passage. After 32 passages no DNA specific for REV was detectable by PCR. Further extensive serological investigations of field sera for antibodies against FPV and REV were carried out. As there was no commercial ELISA available for the detection of antibodies against FPV, an indirect ELISA for this purpose was established. The sensitivity was 85.3 %, the specificity 100 %. Afterwards the repeatability of the obtained results of the ELISA was tested. The intraassay coefficients showed a good repeatability. The interassay coefficients were not satisfying. One explanation was an insufficient stability of the coating antigen when partly used plates were stored at 4°C resp. frozen a second time. In comparison to other methods for detecting antibodies against FPV the ELISA was marginally less sensitive than indirect immunfluorescence and more sensitive than AGP. The investigation of sera from flocks that were exposed to natural infection with FPV for antibodies against FPV and REV showed a proportion of about 50 % with antibodies against FPV and a higher proportion with antibodies against REV. There were some differences between the flocks. A similar proportion of sera from flocks that were vaccinated against FPV had antibodies against FPV, but only in two of ten investigated flocks chickens had antibodies against REV. This makes clear, that the REV antibodies were induced by the integrated fvRP and not by an accidental coinfection with REV. This also shows that most of German FPV strains are able to induce antibodies against REV. Two experimental infection studies were done to investigate the humoral immune response against FPV and REV after intracutaneous infection with different in vitro passages of a field isolate and with the vaccine strain HP B. In both trials the low passaged field isolate with the integrated fvRP induced no antibodies against FPV. The proportion of birds with antibodies against REV varied depending on the infection dose. In contrast to that the vaccine strain induced antibodies against FPV in all birds but not against REV independently from the infecting dose. The highly passaged field isolate, in which the fvRP could not be found by PCR, induced antibodies against FPV only in a few birds and no antibodies against REV. So it was assumed, that the integrated fvRP contributes to the suppression of the antibody response against FPV after an infection with a field isolate. In the first trial the birds were reinfected after five weeks by intravenous application of the low passaged field isolate. About half the birds that previously had been infected with the field isolate developed antibodies against FPV. The proportion of birds with antibodies against REV increased marginally. The birds that had been vaccinated previously showed no antibody response against REV. The reason for this may be that the vaccination prevented the replication of the FPV field isolate and so the development of antibodies. After the intracutaneous infection with the low passaged field isolate and after the intravenous reinfection DNA specific for REV was detected in the peripheral blood mononuclear cells. FPV DNA could only be detected in one of all examined samples. This indicates that infectious REV virions can be formed from the FPV field isolate with integrated fvRP

The effect of diatomaceous earth in live, attenuated infectious bronchitis vaccine, immune responses, and protection against challenge.

Live virus vaccines are commonly used in poultry production, particularly in broilers. Massive application and generation of a protective local mucosal and humoral immunity with no adverse effects is the main goal for this strategy. Live virus vaccines can be improved by adding adjuvants to boost mucosal innate and adaptive responses. In a previous study we showed that diatomaceous earth (DE) can be used as adjuvant in inactivated vaccines. The aim of this study was to test DE as adjuvant in an Ark-DPI live infectious bronchitis virus (IBV) vaccine after ocular or spray application. Titrating the virus alone or after addition of DE showed that DE had no detrimental effect on the vaccine virus. However, adding DE to the vaccine did not induce higher IgG titers in the serum and IgA titers in tears. It also did not affect the frequency of CD4+ T cells, CD8+ T cells and monocytes/macrophages in the blood and the spleen determined by flow cytometry. In addition, protection generated against IBV homologous challenges, measured by viral load in tears, respiratory signs and histopathology in tracheas, did not vary when DE was present in the vaccine formulation. Finally, we confirmed through our observations that Ark vaccines administered by hatchery spray cabinet elicit weaker immune responses and protection against an IBV homologous challenge compared to the same vaccine delivered via ocular route

Referenceable mobile crowdsensing architecture: A healthcare use case

Smartphones have become an integral part in life of users, mainly because over the course of recent years, they have become extremely mainstream, cheap, flexible, and they pack high-end hardware that offers high computational capabilities. Many, if not all of today’s smartphones are equipped with sophisticated sensors which enable smart mobile sensing. The programmable nature of these sensors in the smartphones enable a wide array of possibilities to achieve user-centric or environmental sensing. Even though there have been different approaches proposed to develop a smartphone app, platform, design frameworks, APIs, and even application-specific architectures, there is a lack of generalised referenceable architecture in the literature. In this paper, we propose a generic reference architecture, which can be derived to create more concrete mobile sensing or mobile app architectures. Furthermore, we realise the proposed reference architecture in a healthcare use case, specifically in the context of applying smart mobile sensing to support tinnitus research

Towards Automated Smart Mobile Crowdsensing for Tinnitus Research

Tinnitus is a disorder that is not entirely understood, and many of its correlations are still unknown. On the other hand, smartphones became ubiquitous. Their modern versions provide high computational capabilities, reasonable battery size, and a bunch of embedded high-quality sensors, combined with an accepted user interface and an application ecosystem. For tinnitus, as for many other health problems, there are a number of apps trying to help patients, therapists, and researchers to get insights into personal characteristics but also into scientific correlations as such. In this paper, we present the first approach to an app in this context, called TinnituSense that does automatic sensing of related characteristics and enables correlations to the current condition of the patient by a combined participatory sensing, e.g., a questionnaire. For tinnitus, there is a strong hypothesis that weather conditions have some influence. Our proof-of-concept implementation records weather-related sensor data and correlates them to the standard Tinnitus Handicap Inventory (THI) questionnaire. Thus, TinnituSense enables therapists and researchers to collect evidence for unknown facts, as this is the first opportunity to correlate weather to patient conditions on a larger scale. Our concept as such is limited neither to tinnitus nor to built-in sensors, e.g., in the tinnitus domain, we are experimenting with mobile EEG sensors. TinnituSense is faced with several challenges of which we already solved principle architecture, sensor management, and energy consumption

Towards Incentive Management Mechanisms in the Context of Crowdsensing Technologies based on TrackYourTinnitus Insights

The increased use of mobile devices has led to an improvement in the public health care through participatory interventions. For example, patients were empowered to contribute in treatment processes with the help of mobile crowdsourcing and crowdsensing technologies. However, when using the latter technologies, one prominent challenge constitutes a continuous user engagement. Incentive management techniques can help to tackle this challenge by motivating users through rewards and recognition in exchange of task completion. For this purpose, we aim at developing a conceptual framework that can be integrated with existing mHealth mobile crowdsourcing and crowdsensing platforms. The development of this framework is based on insights we obtained from the TrackYourTinnitus (TYT) mobile crowdsensing platform. TYT, in turn, pursues the goal to reveal insights to the moment-to-moment variability of patients suffering from tinnitus. The work at hands presents evaluated data of TYT and illustrates how the results drive the idea of a conceptual framework for an incentive management in this context. Our results indicate that a proper incentive management should play an important role in the context of any mHealth platform that incorporates the idea of the crowd

Evolution of Magnetic Fields in Stars Across the Upper Main Sequence: II. Observed distribution of the magnetic field geometry

We re-discuss the evolutionary state of upper main sequence magnetic stars using a sample of Ap and Bp stars with accurate Hipparcos parallaxes and definitely determined longitudinal magnetic fields. We confirm our previous results obtained from the study of Ap and Bp stars with accurate measurements of the mean magnetic field modulus and mean quadratic magnetic fields that magnetic stars of mass M < 3 M_sun are concentrated towards the centre of the main-sequence band. In contrast, stars with masses M > 3 M_sun seem to be concentrated closer to the ZAMS. The study of a few known members of nearby open clusters with accurate Hipparcos parallaxes confirms these conclusions. Stronger magnetic fields tend to be found in hotter, younger and more massive stars, as well as in stars with shorter rotation periods. No evidence is found for any loss of angular momentum during the main-sequence life. The magnetic flux remains constant over the stellar life time on the main sequence. An excess of stars with large obliquities beta is detected in both higher and lower mass stars. The obliquity angle distribution as inferred from the distribution of r-values appears random at the time magnetic stars become observable on the H-R diagram. After quite a short time spent on the main sequence, the obliquity angle beta tends to reach values close to either 90 deg or 0 deg for M < 3 M_sun. The evolution of the obliquity angle beta seems to be somewhat different for low and high mass stars. While we find a strong hint for an increase of beta with the elapsed time on the main sequence for stars with M > 3 M_sun, no similar trend is found for stars with M < 3 M_sun. However, the predominance of high values of beta at advanced ages in these stars is notable.Comment: 16 pages, 22 figures, 5 tables, accepted for publication in A

Influence of Different Storage Media, Temperatures and Time Duration on Susceptibility of Ornithobacterium rhinotracheale

Ornithobacterium rhinotracheale (ORT) is an important respiratory pathogen of chickens and turkeys. Isolation of the bacterium from diseased birds is necessary for serotyping, to determine the antimicrobial susceptibility for an effective therapy and to produce autogenous vaccines. A series of experiments was carried out to determine optimal conditions for storage of swabs soaked in ORT suspension. Swabs were immersed in viable ORT suspensions with different bacterial counts and then stored under different conditions. At several time points the viable ORT count in the swabs was determined. Dry cotton swabs as well as three transport media, namely Amies gel medium (AG), Amies gel medium with charcoal (AC), and Stuart gel medium (SG) were tested. ORT could be reisolated from dry swabs stored at room temperature for up to five days and from swabs stored in the media at room temperature for more than seven days. Differences among the transport media were minor. The minimal number of cfu in the ORT-suspension, in which the swabs were soaked, was 105 cfu/ml for successful reisolation of ORT one day post immersion from swabs stored at room temperature in AC medium, and 106 cfu/ml was successful for reisolation from dry swabs. Higher inoculation doses and storage at 4°C prolonged the period in which ORT could be reisolated. Storage of dry swabs at -20°C allowed reisolation of ORT at a constant level for at least 5 d.p.i. Inoculation of swabs with ORT and E. coli reduced the period for which ORT could be reisolated

Smartphone Apps in the Context of Tinnitus: Systematic Review

Smartphones containing sophisticated high-end hardware and offering high computational capabilities at extremely manageable costs have become mainstream and an integral part of users' lives. Widespread adoption of smartphone devices has encouraged the development of many smartphone applications, resulting in a well-established ecosystem, which is easily discoverable and accessible via respective marketplaces of differing mobile platforms. These smartphone applications are no longer exclusively limited to entertainment purposes but are increasingly established in the scientific and medical field. In the context of tinnitus, the ringing in the ear, these smartphone apps range from relief, management, self-help, all the way to interfacing external sensors to better understand the phenomenon. In this paper, we aim to bring forth the smartphone applications in and around tinnitus. Based on the PRISMA guidelines, we systematically analyze and investigate the current state of smartphone apps, that are directly applied in the context of tinnitus. In particular, we explore Google Scholar, CiteSeerX, Microsoft Academics, Semantic Scholar for the identification of scientific contributions. Additionally, we search and explore Google’s Play and Apple's App Stores to identify relevant smartphone apps and their respective properties. This review work gives (1) an up-to-date overview of existing apps, and (2) lists and discusses scientific literature pertaining to the smartphone apps used within the context of tinnitus

Innovations in Doctoral Training and Research on Tinnitus:The European School on Interdisciplinary Tinnitus Research (ESIT) Perspective

Tinnitus is a common medical condition which interfaces many different disciplines, yet it is not a priority for any individual discipline. A change in its scientific understanding and clinical management requires a shift toward multidisciplinary cooperation, not only in research but also in training. The European School for Interdisciplinary Tinnitus research (ESIT) brings together a unique multidisciplinary consortium of clinical practitioners, academic researchers, commercial partners, patient organizations, and public health experts to conduct innovative research and train the next generation of tinnitus researchers. ESIT supports fundamental science and clinical research projects in order to: (1) advancing new treatment solutions for tinnitus, (2) improving existing treatment paradigms, (3) developing innovative research methods, (4) performing genetic studies on, (5) collecting epidemiological data to create new knowledge about prevalence and risk factors, (6) establishing a pan-European data resource. All research projects involve inter-sectoral partnerships through practical training, quite unlike anything that can be offered by any single university alone. Likewise, the postgraduate training curriculum fosters a deep knowledge about tinnitus whilst nurturing transferable competencies in personal qualities and approaches needed to be an effective researcher, knowledge of the standards, requirements and professionalism to do research, and skills to work with others and to ensure the wider impact of research. ESIT is the seed for future generations of creative, entrepreneurial, and innovative researchers, trained to master the upcoming challenges in the tinnitus field, to implement sustained changes in prevention and clinical management of tinnitus, and to shape doctoral education in tinnitus for the future