The genome of hepatitis B virus (HBV) can be assessed by sequence analysis of HBV DNA in serum. In most chronic HBV infected patients treated with potent nucleos(t)ide analogues (NAs) this approach however is limited by the fast decrease of serum HBV DNA during NA treatment. In contrast, HBV RNA was shown to persist in serum of some HBV infected individuals receiving NA treatment. In this study, we established the sequencing of serum HBV RNA as a method for the monitoring of HBV variants during NA treatment after the decrease of serum HBV DNA to undetectable levels. Using this approach, we studied the evolution of HBV variants in follow-up serum samples (n=156) of 25 patients treated with the potent polymerase inhibitor tenofovir (TDF) as second or third-line treatment. In our cohort, specific reverse transcribed full-length and truncated HBV RNA remained detectable with real-time PCR for long periods in most serum samples, also after the decline of HBV DNA during treatment with TDF. The HBV genome could be analyzed based on serum HBV DNA sequencing in 61 serum samples for a mean duration of 6.0 ± 4.5 (0 – 13) months. After this, sequencing of reverse transcribed serum HBV RNA allowed the analysis of the HBV genome for an additional mean duration of 33.9 ± 12.7 (16 - 65) months in 68 serum samples. The comparison of serum HBV DNA and serum HBV RNA derived sequences showed a high homology. In most patients, acquired HBV resistance variants in the reverse transcriptase (rt) region of the polymerase gene were detectable on HBV DNA and HBV RNA basis. Serum HBV RNA sequencing further revealed a long persistence of these variants during TDF treatment (mean duration of 26.5 ± 15.8 (0 – 50) months), which indicates a high conservation in the cccDNA of the infected individuals. Also HBV stop mutations in the small surface (s) gene, which were discussed in the pathogenesis of hepatocellular carcinoma (HCC), were present at baseline in 5 patients and remained detectable on HBV RNA basis during follow-up. In this study, we demonstrated that sequencing of reverse transcribed HBV RNA from patient serum is a suitable method to assess HBV variants during NA treatment. We further provided insights into the evolution of HBV variants during strong suppression of the viral replication with the polymerase inhibitor TDF. Future studies should investigate more comprehensively the clinical application of the here presented method of serum HBV RNA sequencing for the early detection of resistant HBV variants during NA treatment and the observation of HBV s gene variants related to HCC development.:Table of content I Table of content 3 II Abbreviations 6 1 Introduction 10 1.1 HBV 11 1.1.1 Classification 11 1.1.2 HBV virion structure and genomic organization 11 1.1.3 HBV proteins 12 1.1.4 HBV replication cycle 15 1.2 Chronic HBV infection 17 1.2.1 Epidemiology 17 1.2.2 Natural course of chronic HBV infection 17 1.3 Treatment of chronic HBV infection with nucleos(t)ide analogues 18 1.3.1 Nucleos(t)ide analogues 18 1.3.2 Treatment goals 18 1.3.3 Response to nucleos(t)ide analogue treatment 20 1.3.4 Treatment with nucleos(t)ide analogues and liver disease 21 1.4 Evolution of HBV variants during antiviral treatment 22 1.4.1 HBV resistance mutations in the pol gene 22 1.4.2 Resistance rates to treatment with nucleos(t)ide analogues 25 1.4.3 HBV variants in the s gene 26 1.5 HBV RNA in serum of chronically infected patients 29 1.5.1 HBV RNA molecules 29 1.5.2 HBV RNA packaging and release 29 1.5.3 HBV RNA as serum marker 31 1.6 Aim of the study 31 2 Materials and methods 33 2.1 Materials 33 2.1.1 Chemicals 33 2.1.2 Devices 33 2.1.3 Laboratory materials 34 2.1.4 Cycler 34 2.1.5 Kits 35 2.1.6 Buffers and solutions 36 2.1.7 Primers 36 2.1.8 Data analysis 39 2.2 Methods 39 2.2.1 Patient set and sample selection 39 2.2.2 Extraction of nucleic acids from serum samples 40 2.2.3 Reverse transcription of HBV RNA 40 2.2.4 Quantification of HBV serum DNA and HBV RNA by real-time PCR 41 2.2.4.1 Real-time PCR 41 2.2.4.2 Quantification of serum HBV DNA 43 2.2.4.3 Quantification of serum HBV trRNA and HBV flRNA 45 2.2.5 Sequencing of serum HBV DNA and HBV RNA 47 2.2.5.1 Primer design 47 2.2.5.2 Amplification by PCR 48 2.2.5.3 Purification of amplification products 50 2.2.5.4 Sanger sequencing of PCR fragments 51 2.2.6 Quantification of serum HBsAg and HBeAg 53 2.2.7 Cloning of HBV variants 53 2.2.8 Data analysis 55 2.2.8.1 Serum HBV DNA and HBV RNA quantities 55 2.2.8.2 Analysis of HBV DNA and HBV RNA sequences 55 3 Results 59 3.1 Composition of the patient set 59 3.2 Quantification of HBV DNA and HBV RNA in serum samples 59 3.2.1 Quantitative courses of serum HBV DNA 60 3.2.2 Quantitative courses of serum HBV flRNA and HBV trRNA 61 3.3 Sequencing of HBV DNA and HBV RNA 64 3.3.1 Method 64 3.3.2 Follow-up with sequencing of HBV DNA and HBV RNA 67 3.3.3 Genotyping of baseline samples 67 3.4 Evolution of HBV variants in the rt region 68 3.4.1 HBV resistance mutations in the rt region at baseline 68 3.4.2 HBV resistance mutations in the rt region during antiviral treatment 70 3.5 HBV variants in the s gene 77 3.5.1 HBV s gene variants at baseline 77 3.5.2 HBV s gene variants during antiviral treatment 80 4 Discussion 82 4.1 Patient cohort 82 4.2 Quantification of serum HBV DNA, HBV flRNA and HBV trRNA 82 4.3 Quantitative courses of serum HBV DNA, HBV flRNA and HBV trRNA 83 4.4 Sequencing of serum HBV RNA as novel method for HBV genome analysis 85 4.5 Evolution of HBV variants in the rt region 89 4.6 Evolution of HBV stop mutations in the s gene 91 4.7 Conclusion 92 III Summary 94 IV References 96 V List of Figures 104 VI List of Tables 105 VII Supplement 106 VIII Erklärung über die eigenständige Abfassung der Arbeit 107 IX Curriculum Vitae 108 X Publications 110 XI Acknowledgment 114

Die entzündliche Kardiomyopathie ist eine heterogene Erkrankung. Die häufigste Ursache ist eine Virusinfektion, wobei Parvovirus B19 (B19V) der bedeutendste Erreger ist. In dieser Arbeit wurden potenzielle Therapie für die speziellen klinischen Verläufe und deren Phänotypen untersucht. In vitro konnte gezeigt werden, dass Telbivudin in B19V-infizierten/B19V-non-structural protein-1-stimulierten humanen mikrovaskulären Endothelzellen (HMEC-1) endothelial-protektiv wirkt. In einem klinischen Versuch wurden dann 4 Patienten, bei denen eine aktive Transkription des B19V nachgewiesen wurde, für 6 Monate mit Telbivudin behandelt. Alle Patienten verbesserten sich. Ein anderes klinisches Szenario stellt die schwere Entzündung des Myokards dar, welche gewöhnlich mit einer inaktiven/persistierenden Infektion des B19V verbunden ist. Eine Behandlung mit Immunsuppressiva ist hier umstritten, da eine Reaktivierung des Virus befürchtet wird. Um diesen Aspekt weiter zu untersuchen, würde eine Therapie mit Prednisolon in Kombination mit Azathioprin bei 51 B19V-positiven und 17 B19V-negativen Patienten angewandt. Beide Gruppen profitierten in ähnlichem Maße von der Kombinationstherapie, wobei sich die Virusmenge nicht signifikant veränderte. Bei B19V-negativen Patienten konnte über die Persistenz von CD20+ B-Lymphozyten in den EMBs die Untergruppe der „Steroide non-responder“ klassifiziert werden. Im weiteren Verlauf wurden 6 Patienten mit Rituximab, einem monoklonalen Antikörper, der spezifisch gegen CD20+ B-Lymphozyten gerichtet ist, behandelt. Hiervon zeigten 5 Patienten eine ausgezeichnete klinische Verbesserung. Ein Patient mit Myokarditis-induzierten kardiogenen Schock zeigt, dass die Entlastung des linken Ventrikels mittels eines Mikroaxialpumpensystems zu einer rapiden Abnahme der Entzündungszellen führt. Zusammenfassend liefert diese Arbeit Belege für die Wirksamkeit und die Notwendigkeit einer phänotypbasierten Behandlung bei der entzündlichen Kardiomyopathie. / Inflammatory cardiomyopathy is a heterogenous disease. Viral etiologies are the most common, with parvovirus B19 (B19V) being the most prominent culprit. Currently, no specific treatment for inflammatory cardiomyopathy exists. In this study, tailored treatment strategies were investigated as potential therapies for specific clinical scenarios. The antiviral drug telbivudine was investigated in the setting of EMB-proven B19V-associated inflammatory cardiomyopathy. In cell culture, telbivudine exhibited endothelial-protective effects on B19V-infected/B19V-non-structural protein-1-stimulated human microvascular endothelial cells (HMEC-1). Clinically, four B19V-positive patients improved following six-month telbivudine regimen in a single-patient use approach. The results were translated to the “PreTOPIC” clinical study, for further evaluation in a randomized placebo-controlled setting. In a different clinical scenario, severe myocardial inflammation is usually associated with inactive/persistent B19V. Here, the use of immunosuppression is controversial, fearing viral flare-up. We investigated combined prednisolone/azathioprine therapy in 51 B19V-positive and 17 B19V negative patients in a single-center observational study. Both groups gained similar benefit, while viral loads did not significantly vary. Among virus-negative phenotypes, EMB-proven CD20+ B lymphocyte persistence characterized a subgroup of steroid non-responders. In this cohort, six patients were treated with rituximab, a monoclonal antibody selectively targeting CD20+ B lymphocytes. Five patients showed outstanding clinical improvement parallel to CD20+ B lymphocyte depletion. Lastly, in a single case of myocarditis-induced cardiogenic shock, mechanical left ventricular unloading via axial flow pump proved to exert disease-modifying effects. In conclusion, this thesis provides evidence for the efficacy and need for phenotype-based inflammatory cardiomyopathy treatment.

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