Recent Advancement in Hepatitis B Virus, Epigenetics Alterations and Related Complications

Worldwide, it is estimated that more than 400 million people are currently living with chronic hepatitis B virus (HBV) infection, contributing to more than one million deaths annually as a result of liver cirrhosis and hepatocellular carcinoma (HCC). HBV DNA integrates into the cellular DNA in liver tissue of patients with chronic HBV infection and HCC. Following HBV infection, DNA methyltransferases (DNMTs) methylate any HBV DNA integrated into the human genome. This novel epigenetic mechanism enables the suppression of HBV antigens, leading to reduced viral replication. HBV is thought to induce DNA methylation via hepatitis B x (HBx) protein, which modulates cellular signalling pathways by activating DNMT 1 and 3 to benefit the virus. Activation of DNMT 1 and 3 inappropriately methylates host cellular genes including tumour suppressor genes whose disruption causes transformation of hepatocytes and hepatic malignancy. By being localised in the cytoplasm, nucleus and mitochondria of HBV-infected hepatocytes, it appears that HBx protein manages to exploit the entire body of cellular signalling pathways for viral survival and propagation. HBx protein may achieve its transcriptional transactivation action by either interacting with key genes or altering their related cellular signalling pathways or by hijacking their binding partners and taking over their roles. Although the underlying mechanisms are still unclear, processes such as cell cycle progression, calcium homeostasis, hepatic metabolism, protein ubiquitination, RNA splicing and vitamin D receptor regulation are key mechanisms that HBx protein alters to favour viral replication and cell survival. These detrimental effects would connect HBV infection to malignant transformation by inducing uncontrolled cell growth, proliferation and disrupting apoptosis

Hepatitis E virus superinfection and human SOCS3 and ISG15 in hepatitis B virus-related liver diseases

Although the introduction of the Hepatitis B virus (HBV) vaccine has significantly reduced mortality and morbidity in many parts of the world, few pockets of high endemicity still remains in South-East Asia and in Sub-Saharan Africa. Despite, Vietnam initiated universal immunization for hepatitis B for infants in 2003, the rates of chronically infected patients with hepatitis B remains high with an estimated prevalence of >10%. The clinical course of HBV infection is influenced by both host and viral factors. In this thesis, I utilized a cohort of patients well characterized for clinical HBV infections, including acute and chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma. In chapter one of my thesis, I investigated 1318 Vietnamese HBV patients to elucidate if superinfection by other hepatitis viruses exists in this study population. In particular, I studied the HEV seroprevalences in patients with HBV and characterized specific HEV isolates at the molecular level to understand the consequences of HEV superinfections on the clinical course of HBV infections. This study showed that HEV may aggravate the clinical outcome of HBV infection. In chapter two of my thesis, I studied the contribution of negative regulator suppressor of cytokine signaling-3 (SOCS3) promoter variants in HBV disease. I genotyped 878 HBV patients and 272 healthy controls for SOCS3 promoter variants. SOCS3 promoter hyper methylation in HBV tumor tissues was examined by bisulfite sequencing and mRNA expression was quantified in tumor and non-tumor tissues. The results revealed that SOCS3 promoter variants are associated with HBV susceptibility and SOCS3 hypermethylation stimulates HCC development. Additionally in chapter 2, investigations were carried out to associate Interferonstimulated gene 15 (ISG15) polymorphisms, ISG15 serum levels, and relative mRNA expression with HBV-related liver diseases. ISG15 exon2 variant rs1921 was associated with HBV susceptibility. ISG15 serum levels were higher among HBV patients and were positively correlated with HBV-DNA loads. ISG15 mRNA expression was increased in HBV patients. The results reveal that ISG15 appears to be a proviral factor involved in HBV replication and triggers the progression of HBVrelated liver diseases. Taken together, this dissertation serves as a basis to understand the association of host and viral factors with HBV susceptibility and subsequent clinical outcomes of HBV related liver diseases.Trotz der Einführung eines effektiven Hepatitis-B-Virus (HBV) Impfstoffs, der weltweit die Mortalität und Morbidität einer HBV-Infektion signifikant reduziert hat, gibt es dennoch einige Regionen, insbesondere Südostasien und Sub-Sahara Afrika, in denen HBV-Infektionen hoch endemisch sind. Obwohl Vietnam im Jahr 2003 eine generelle Immunisierung gegen Hepatitis B für Säuglinge und Jugendliche eingeführt hat, ist in diesem Land die Rate von chronisch HBV-Infizierten mit einer geschätzten HBV-Prävalenz von >10% noch immer hoch. Bekannt ist, dass der klinische Verlauf einer HBV Infektion sowohl von Wirt- als auch viralen Faktoren beeinflusst wird. Um dies weiter zu untersuchen wurde in der vorliegenden Doktorarbeit Proben von einer gut charakterisierten Kohorte von Patienten mit HBVInfektion eingesetzt, die Patienten mit akuter und chronischer Hepatitis B, Leberzirrhose und hepatozellulärem Karzinom einschloss. In Kapitel 1 der vorliegenden Dissertation wurden Proben von 1318 vietnamesischen HBV infizierten Patienten untersucht, um zu klären, ob Superinfektionen mit anderen Hepatitisviren in dieser Studienpopulation auftreten. Im Einzelnen wurde die Hepatitis E Virus (HEV) Seroprävalenz in den HBV-positiven Patienten analysiert. Spezifische HEV-Stränge wurden weiter molekular-genetisch charakterisiert um die Auswirkung einer HEV-Superinfektion auf den klinischen Verlauf einer HBVInfektion besser zu verstehen. Diese Studie zeigte, dass HEV den klinischen Verlauf einer HBV-Infektion verschlimmern kann. In Kapitel 2 dieser Arbeit wurde der Beitrag von negative regulator suppressor of cytokine signaling-3 (SOCS3) Promotorvarianten auf den Verlauf einer HBV-Infektion analysiert. Dazu wurden SOCS3 Promotorvarianten, isoliert aus 878 HBV-positive Patienten und 272 gesunden Kontrollindividuen, näher charakterisiert. SOCS3 Promotor- Hypermethylierung in Tumorgewebe von HBV-positiven Patienten wurde mittels Bi-Sulfit Sequenzierung analysiert und zudem die SOCS3 mRNA-Expression in Tumor und Nicht-Tumorgewebe dieser Patienten quantifiziert. Die Ergebnisse dieser Untersuchungen zeigten, dass SOCS3 Promotorvarianten mit der HBV Suszeptibilität assoziiert waren und SOCS3 Promotor-Hypermethylierung die HCC Entwicklung stimulierte. Zusätzlich dazu wurde in Kapitel 2 weiterführende Experimente durchgeführt, die eine Assoziation von Interferon-stimulated gene 15 (ISG15)-Polymorphismen, ISG15 Serumspiegel und relativer ISG15 mRNA Expression mit HBV-bedingten Leberentzündungen aufdecken sollten. Hierbei zeigte sich, dass die ISG15 exon2 Variante rs1921 mit der HBV-Suszeptibilität assoziiert war und ISG15 Serumspiegel in HBV-positiven Patienten höher und positiv korreliert mit der HBV-DNA Viruslast waren. Zudem war die ISG15 mRNA Expression in HBV-positiven Patienten erhöht. Die Ergebnisse belegten, dass ISG15 ein proviraler Faktor ist, der bei die HBV-Replikationskontrolle involviert ist und die Progression HBV-bedingter Leberentzündungen triggert. Zusammenfassend bildet die vorliegende Dissertation eine Grundlage für das bessere Verständnis essentieller Assoziationen von Wirt- und viralen Faktoren mit der HBV Suszeptibilität und folgend dem klinischen Verlauf HBV-bedingter Lebererkrankungen

An investigation of genome-wide promoter region cytosine-phosphate-guanine (CpG) Island methylation profiles in patients with chronic hepatitis B virus infection

Includes bibliographical references.Hepatitis B virus (HBV) is oncogenic and a major cause of hepatocellular carcinoma (HCC) in the developing world. It integrates parts of its genome such as the HBx gene, core and surface antigens into the human genome. The integrated viral DNA disrupts gene function resulting in physiological changes that cause liver disease. The viral inserts are inactivated through methylation. This is a protective innate response driven by human DNA methyltransferases triggered by the presence of viral DNA inserts. This thesis investigates the hypothesis that during the innate response to methylate integrated HBV DNA, there is unintended methylation of genomic DNA around the intercalated viral DNA that could be adjacent host promoter Cytosine-phosphateGuanine (CpG) islands. This would activate or silence genes including tumour suppressors and result in the clinical disease phenotypes of hepatic inflammation, fibrosis and HCC that characterise chronic HBV infection. Genome-wide microarray analysis was used to investigate for the presence of promoter CpG island methylation in a cohort of patients with liver disease due to HBV infection, HCC, autoimmune hepatitis which is a non-viral liver disease and normal cases with no liver disease. The study identified hypermethylation in promoter regions, transcription start sites, gene exons and introns. Only sites in the promoter region and within 100bp upstream of a transcription start site were analysed for this thesis presentation. Using an extended cohort of patients with chronic HBV infection and normal controls, bisulfite DNA sequencing was used to validate and confirm the presence of DNA methylation in a selection of some of genes identified. HBV infected patients were shown to have hypermethylation in the promoter CpG island regions of several genes that regulate hepatic metabolism, tumour suppression, ribonucleic acid splicing, vitamin D receptor binding, protein ubiquitination and the cell cycle. Many of these genes have transcriptional binding factors that are known to be affected by the transcriptional transactivator HBx protein, suggesting that HBx protein is important in the pathogenesis of liver disease. Amongst the most hypermethylated core promoter regions identified were those for cyclin kinases genes such as Cyclin D3 (CCND3). CCND3 gene is important in liver regeneration and wound healing and its abnormal function has been linked to the development of liver fibrosis and HCC. Increased methylation of CCND3 gene was associated with HBV e antigen positive status and genotype D, supporting the hypothesis that increased methylation is associated with host and viral factors. Methylation induced alteration in the function of the identified gene promoters would affect cellular signalling with effects on cell growth, differentiation, proliferation and apoptosis. These changes would explain the development of hepatic inflammation, apoptosis, fibrosis and malignant transformation seen in chronic HBV infection. Further investigation of these genes will provide new insights on mechanisms of HBV induced liver disease and the development of new molecular diagnostic tools or therapeutic interventions

RASSF1A and p16 promoter methylation and treatment response in chronic hepatitis C genotype 1b patients treated with pegylated interferon/ribavirin

Prevention of chronic hepatitis C (CHC) and its complications is based on antiviral therapy and early detection of reliable molecular markers in persons under risk. We investigated whether the methylation status of RASSF1A and p16 genes, alone or in combination with host and viral factors, affects the response to therapy with pegylated interferon/ribavirin (PEG-IFN/RBV). Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of the target promoter sequences of RASSF1A and p16 in circulating-free DNA from the peripheral blood of 49 patients with CHC genotype 1b. The methylation status of the examined genes did not affect the response to therapy. However, the simultaneous presence of either RASSF1A or p16 methylation and the CC genotype of IL28B was significantly related to a sustained virologic response (P=0.009 and P=0.032, respectively). After Bonferroni correction, only the result concerning the RASSF1A gene remained significant (P<0.0125). Methylation of RASSF1A was associated with the CC genotype of the IL28B gene (P=0.024) and a higher viral load (≥400 000 IU/mL, P=0.009). Our results suggest that combined analysis of RASSF1A gene methylation and IL28B rs12979860 polymorphism could potentially help in the prediction of therapy response in CHC genotype 1b patients

Estudio de la capacidad adyuvante del plásmido que contiene la secuencia CpG específica de cerdos en dos vacunas porcinas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Sanidad Animal, leída el 22-07-2013Depto. de Sanidad AnimalFac. de VeterinariaTRUEunpu

Mapping a Gene Responsible for Natural Resistance to Rift Valley Fever Virus in Inbred Rats

The Rift Valley Fever virus (RVFV) presents an epidemic and epizootic threat in sub-Saharan Africa, Egypt, and the Arabian Peninsula, and has recently gained attention as a potential weapon of bioterrorism due to its ability to infect both livestock and humans. Inbred rat strains show similar characteristic responses to the disease as humans and livestock, making them a suitable model species. Previous studies had shown differences among various inbred rat strains in susceptibility to RVFV hepatic disease, including a higher susceptibility of Wistar-Furth (WF) rats compared to a more resistant Lewis (LEW) strain. Further study revealed that this resistance trait follows the pattern of a dominant gene inherited in Mendelian fashion. A congenic WF.LEW strain resistant to infection with RVFV was derived from the susceptible WF and resistant LEW strains, and a subsequent genome scan revealed two prospective regions for the location of the gene, one on chromosome 3 and the other on chromosome 9. Subsequently, this study employed the methods of backcrossing, genotyping, viral challenges, gene expression studies, and sequencing to define a practicable region of interest and to further identify a viable candidate gene and prospective mechanism by which resistance is conferred. A program of backcrossing WF.LEW rats to WF rats, genotyping offspring using SNPs and microsatellites, and subsequently challenging N1 litters with RVFV was used to determine that the ~2Mb region on the distal end of chromosome 3 contains the gene conferring resistance. The use of genetic markers to detect recombination in further backcross generations resulted in the identification of two recombinants in this newly established region of interest. Through RVFV challenges, the recombinants narrowed the prospective region of chromosome 3 to ~500Kb containing 20 genes. Comparative qPCR analysis of all 20 genes combined with comparative sequencing studies of the entire region between susceptible WF/NHsd rats and resistant WF.LEW rats facilitated the identification of candidate gene Rtel1 and a proposed mechanism by which resistance is conferred, which will potentially become the basis for developing new preventive measures against the virus

The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses

DNA methylation markers in the detection of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and has a poor prognosis. Epigenetic modification has been shown to be deregulated during HCC development by dramatically impacting the differentiation, proliferation, and function of cells. One important epigenetic modification is DNA methylation during which methyl groups are added to cytosines without changing the DNA sequence itself. Studies found that methylated DNA markers can be specific for detection of HCC. On the basis of these findings, the utility of methylated DNA markers as novel biomarkers for early-stage HCC has been measured in blood, and indeed superior sensitivity and specificity have been found in several studies when compared to current surveillance methods. However, a variety of factors currently limit the immediate application of these exciting biomarkers. In this review, we provide a detailed rationalisation of the approach and basis for the use of methylation biomarkers for HCC detection and summarise recent studies on methylated DNA markers in HCC focusing on the importance of the aetiological cause of liver disease in the mechanisms leading to cancer.</p

Molecular evolution and origins of hepatitis B virus in humans and non-human primates

Infection with hepatitis B virus (HBV) has been detected in most populations throughout the world, as well as in a number of non-human primate species. In humans HBV infection represents a major global health problem, with an estimated 1 million deaths per year due to hepatocellular carcinoma and chronic hepatitis. HBV variants infecting humans can be classified into at least 7 different genotypes differing from each other by 11-13% in nucleotide sequences. A range of distinct genotypes also infect African apes, Asian apes and possibly New World monkeys. Studies of HBV epidemiology and the geographical species associations of different HBV genotypes have led to a number of hypotheses for the origin of HBV in humans and primates. These are the "Out of Africa" hypothesis, the hypothesis that HBV originated in South America and spread in Africa and Western countries in the last 200-300 years, and more recently, proposed origins from cross-species transmission and/or co-evolution of HBV in their current host. The main aim of this thesis is to investigate the molecular evolution of human and non-human primate HBV to gain further insights into the origin of HBV in these species. This investigation was carried out in three main sections.The first comprised an extensive and detailed genetic analysis of the distribution of human HBV genotypes in HBV endemic areas in sub-Saharan Africa and South East Asia. In the second section complete genome sequences of HBV variants were analysed for recombination between different HBV genotypes. This analysis included the use of a novel method based on the calculation of association scores for phylogenetic groups, an approach that helps resolve many of the uncertainties and difficulties of interpretation of results arising from conventional methods, such as SimPlot.The third section investigated the frequencies of HBV infection in non-human primates, and the relationship between HBV genotype, primate species and geographical range. In my survey, HBV infection was confined to African and Asian apes, and uniformly absent from a wide range of African monkey species. Phylogenetic analysis of chimpanzee-, gibbon- and orangutan-derived HBV variants indicated that a geographical rather than a species correlation with genotypes, implying the co-circulation and cross-species transmission of HBV between species of overlapping habitats. However, in no cases were primate-associated HBV variants found in humans, nor human genotypes in non-human primates. These findings and the interspersed nature of human and non-human primate HBV genotypes deepens the mystery of HBV origins and evolution in humans. The findings, however, provide a context for ongoing studies of HBV biological variability and genotype-associated differences in pathogenicity and outcomes of infection

Expression analysis of liver-specific circulating micrornas in hcv-induced hepatocellular carcinoma in Egyptian patients

Introduction: The prevalence of hepatocellular carcinoma (HCC) in Africa is higher compared to the rest of the world due to the high incidence of chronic infection with hepatitis C virus (HCV). In Egypt, HCV infection is the leading cause for the high HCC incidence, which is usually diagnosed at late stages. Due to the absence of reliable and accurate biomarkers for early detection of liver cancer, circulating microRNAs have recently emerged as great candidates for early diagnosis of HCC. These small non-coding RNA molecules are responsible for regulating gene expression and RNA stability. Therefore, the aim of this study is to investigate the potential of liver-specific circulating microRNAs as an accurate non-invasive diagnostic tool for the early detection of HCV-induced HCC. Methods: Eight main miRNAs (miR-16, miR-34a, miR-122a, miR-125a, miR-139, miR-145, miR-199a, and miR-221) were selected due to their expression patterns in HCC as well as their contribution to the development of hepato-carcinogenesis. A total of 165 patients were enrolled in this study, from which serum samples were collected and categorized into four main patient groups: 42 chronic hepatitis C (CHC) without cirrhosis, 45 CHC with cirrhosis (LC), 38 HCC with HCV patients, and 40 healthy controls. The expression profile of the eight miRNAs was analyzed using TaqMan real-time reverse transcription-polymerase chain reaction. Additionally, the conventional markers for HCC α-fetoprotein (AFP) and des-γ-carboxyprothrombin (DCP) were measured using commercial kits. Results: Serum levels of miRNA-122a, miRNA-125a, miRNA-139, miRNA-145 and miRNA-199a were significantly lower (p\u3c0.01) in HCC than in both CHC and LC groups. On the other hand, no significant difference was shown in the expression of miR-16, miR-34a, and miR-221 between the CHC, LC, and HCC groups. MiR-16, miR-34a, and miR-221 were significantly elevated in the HCC group compared to the control group. MiR-122a showed the highest specificity and sensitivity, followed by miR-125a, which had the second highest specificity, indicating its significance in diagnosis. Conclusions: The results indicated that measurement of serum levels of miR-122a, miR-125a, miR-139, miR-145, and miR-199a can help to differentiate HCC from CHC and LC. Measurement of serum levels of miR-16, miR-34a, and miR-221 were shown to have a prognostic value. Highly significant correlation was established between different miRNAs within the same patient group or between two different groups, indicating a great diagnostic value for the early detection of HCC. MiR-122a had the highest specificity and sensitivity, indicating that serum miR-122a might serve as a novel and potential non-invasive biomarker for HCV-induced HCC