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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:16946 |
| Date | 04 January 2018 |
| Creators | Schmalbrock, Laura Katharina |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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