MicrobiologyBytes

Biologists have uncovered virus fragments from the same family of the modern Hepatitis B virus locked inside the genomes of songbirds such as the modern-day zebra finch. This article marks the first time that endogenous hepadnaviruses have been found in any organism. An endogenous virus is one that deposits itself or fragments of itself into the chromosome of an organism, allowing it to be passed from generation-to-generation. Previously, most of these known “fossilized” virus sequences have come from retroviruses. These fragments have been sitting in the bird’s genomes for at least 19 million years, far longer than anyone previously thought this family of viruses had been in existence.

The researchers dated the hepadnavirus fragments by locating them in the same spot on the genome of five species of passerine birds and then tracing those species to a common ancestor that lived more than 19 million years ago. This work provides a glimpse into an ancient viral world that we never knew existed. The results are remarkable – hepadnaviruses, and likely many other viruses as well, are far older than we previously thought. Another surprise is that the older versions of the hepadnaviruses are remarkably similar to today’s viruses. This suggests that the slow evolution of the hepadnaviruses observed in birds indicates that the viruses are, in the long run, better adapted to their hosts than what is suggested by study of the disease-causing Hepatitis B viruses. Genomic fossils like these remarkable hepadnaviral fossils have the prospect of completely revising our preconceived notions about the age and evolution of such viruses.

This study also opens new avenues for research that might help predict and prevent human viral pandemics originating in bird species. Given that they were infected in the past, it is legitimate to think that some of these birds may still carry such viruses today. We can use this discovery as a guide to screen targeted groups of bird species for the presence of new circulating Hepatitis B-like viruses.

Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses. (2010) PLoS Biol 8(9): e1000495. doi:10.1371/journal.pbio.1000495
Because most extant viruses mutate rapidly and lack a true fossil record, their deep evolution and long-term substitution rates remain poorly understood. In addition to retroviruses, which rely on chromosomal integration for their replication, many other viruses replicate in the nucleus of their host’s cells and are therefore prone to endogenization, a process that involves integration of viral DNA into the host’s germline genome followed by long-term vertical inheritance. Such endogenous viruses are highly valuable as they provide a molecular fossil record of past viral invasions, which may be used to decipher the origins and long-term evolutionary characteristics of modern pathogenic viruses. Hepadnaviruses (Hepadnaviridae) are a family of small, partially double-stranded DNA viruses that include hepatitis B viruses. Here we report the discovery of endogenous hepadnaviruses in the genome of the zebra finch. We used a combination of cross-species analysis of orthologous insertions, molecular dating, and phylogenetic analyses to demonstrate that hepadnaviruses infiltrated repeatedly the germline genome of passerine birds. We provide evidence that some of the avian hepadnavirus integration events are at least 19 My old, which reveals a much deeper ancestry of Hepadnaviridae than could be inferred based on the coalescence times of modern hepadnaviruses. Furthermore, the remarkable sequence similarity between endogenous and extant avian hepadnaviruses (up to 75% identity) suggests that long-term substitution rates for these viruses are on the order of 10^-8 substitutions per site per year, which is a 1,000-fold slower than short-term rates estimated based on the sequences of circulating hepadnaviruses. Together, these results imply a drastic shift in our understanding of the time scale of hepadnavirus evolution, and suggest that the rapid evolutionary dynamics characterizing modern avian hepadnaviruses do not reflect their mode of evolution on a deep time scale.

Related:

1. Phoenix from the ashes: The 5 million year old virus
2. The Island of Fossil Viruses

Hepatocarcinogenesis (liver cancer) is, and will continue to be a major worldwide health problem. With chronic HBV and HCV infections being responsible for a significant proportion of HCC cases, the development of new and relevant cell culture and animal models to study the interactions of HBV and HCV with their host and the development of efficient means to combat chronic infections will remain major tasks to tackle. This publication gives an overview of our current state of knowledge in respect to the basic biology of these viruses, as well as the clinical and therapeutic options that have been, and are being developed, and highlights the major current technical and biological limitations that the field needs to overcome.

Hepatitis B and C Viruses and Hepatocellular Carcinoma. (2010) Viruses 2(8): 1504-1509 doi:10.3390/v2081504

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• Hepatitis B virus integration and hepatocarcinogenesis
• The search for infectious causes of human cancers
• Liver flukes and cancer

Hepatitis B virus (HBV) infection is a major health problem, with
400 million people chronically infected worldwide who are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). The epidemiological evidence linking HBV infection to HCC is very strong, and despite the mechanisms underlying HBV-associated carcinogenesis remain to be fully defined, a growing number of studies support a direct role of HBV in the process. The HBV-encoded regulatory protein hepatitis B virus X protein (HBx) is thought to contribute to HBV oncogenicity. HBx transforms SV40-immortalized mouse hepatocytes, induces cell cycle progression within the regenerating liver, causes liver cancer in some transgenic mice, and acts as a cofactor to accelerate cancer development in other mouse models. HBx is a 154-amino acid protein with an N-terminal negative regulatory domain and C-terminal transactivation or coactivation domain that has been detected both in the cytoplasm and in the nuclei of infected hepatocytes. Studies in transfected cells have shown that HBx expression affects cellular functions such as cytoplasmic calcium regulation, cell signaling, transcription, cell proliferation, DNA repair, and apoptosis.

The lack of homology of the X ORF to host protein and its high conservation to other mammalian hepadnaviruses genomes strongly suggest that HBx play a role in virus life cycle. Although initial studies suggested that HBx was not required for virus replication in cell culture, experiments with the highly related woodchuck hepatitis virus (WHV) system indicate that the WHV X protein (WHx) is required for virus replication in vivo. Studies performed using a plasmid-based replication assays that use greater-than-unit-length HBV genomes transfected in HCC cell lines or injected via the mouse tail vein have repeatedly confirmed that HBx potentiate HBV replication. The HBx protein behaves as a promiscuous transactivator of viral and cellular promoters. Although the subcellular localization of HBx seems to be mainly cytoplasmic, a small variable fraction of the protein can be found in the nucleus, and the ability of HBx to activate transcription of host genes is thought to occur indirectly by interaction with nuclear transcription factors or by activation of different signal transduction pathways in the cytoplasm.

Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. PNAS USA November 11 2009. doi: 10.1073/pnas.0908365106
HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.

Related:

• Hepatitis B and C viruses
• Hepatitis B virus integration and hepatocarcinogenesis

Chronic hepatitis B virus (HBV) infection affects about 400 million people around the globe, being one of the most common infectious diseases and among the world’s leading causes of death. Antiviral therapy of chronic hepatitis B (CHB) aims to improve quality of life and survival chance of the patients by preventing progression of liver damage to cirrhosis, end-stage liver disease and liver cancer (HCC), thus preventing anticipated liver-related death. This goal is achieved by suppression of HBV replication in a sustained or maintained manner, either by short-term “curative” treatment with standard (IFN) and pegylated interferon (Peg-IFN) or long-term “suppressive” therapy with nucleos(t)ide analogues, like lamivudine, adefovir, entecavir, telbivudine and tenofovir. Since both strategies have advantages and disadvantages, the wise treatment of a patient with CHB requires careful balance between prediction of the natural history of HBV and of the potential benefit of anti- HBV therapy. Recent data on the long-term efficacy of third generation of nucleos(t)ide analogues entecavir and tenofovir have tipped the balance towards long-term suppression therapy as the first-line option for most patients with CHB, independent of the HBeAg status.

Chronic hepatitis C is a major worldwide health problem with an estimated prevalence of 1.6-2%. In Europe, more than 9 million chronic carriers and approximately 86,000 deaths per year are estimated due to the late complications of hepatitis C virus (HCV). The prognosis of chronic hepatitis C depends on the rate of fibrosis progression, which over a 20-30 year time span, may determine the risk of developing cirrhosis and its complications, namely HCC, liver decompensation, hepatic encephalopathy and oesophageal variceal bleeding. The only therapeutic intervention able to halt this progressive process is eradication of HCV by Interferon (IFN)-based therapies. Since the empirical choice to use IFN in 1986, therapy for chronic hepatitis C has constantly evolved over the past decade, with the attainable sustained virological response (SVR) rates increasing through the years. The addition of the guanosine nucleoside analogue ribavirin (Rbv) to IFN can be considered the major breakthrough in the treatment of chronic hepatitis C. Through mechanisms of action that still remain largely unknown, Rbv has determined a greater number of patients to ultimately achieve a SVR by increasing the rates of on-treatment response and reducing the rates of post-treatment relapse. In the large phase III clinical trials designed to assess its efficacy and safety, the combination of IFN and Rbv resulted in SVR rates of 30-35% in HCV genotype 1 patients and 75-80% in HCV-2 and 3 patients. These figures exceeded by far those obtained by IFN monotherapy, effectively leading the way for combination therapy to become the standard of care in the late 1990’s. The latest innovation in the treatment of chronic hepatitis C has been the pegylation of the IFN molecule (PegIFN) through the attachment of one or more polyethylene glycols to the IFN, a process that is able to modify the immunological, pharmacokinetic and pharmacodynamic properties of the drug. Standard IFN was in fact characterized by a number of limitations, such as poor stability, short elimination half-life and potential immunogenicity, that ultimately determined its small antiviral effect. Moreover, due to the increase in elimination half-life obtained by the pegylation process, it has been possible to lengthen the dosing interval from the unpractical three times a week schedule required by standard IFN, to the more “user friendly” once a week administration, a feature that has increased convenience whilst facilitating adherence to the recommended treatment schedule. Following the demonstration of a more potent antiviral effect in terms of SVR rates in phase III randomized trials, PegIFN has become the standard of care for chronic hepatitis C.

One year of interferon therapy inhibits HBV replication in one third of the patients whereas long-term administration of oral nucleos(t)ide analogues is efficient in most of them, as long as early treatment adaptation in patients with partial virological response and resistance is provided. Following the demonstration of a more potent antiviral effect in terms of sustained virological response (SVR) rates, Pegylated-IFN coupled with Ribavirin has become the standard treatment for chronic hepatitis C, with nearly 65% of all treated patients achieving a SVR. Long-term suppression of HBV and eradication of HCV would halt the progression of chronic hepatitis to cirrhosis, hepatocellular carcinoma and liver decompensation.

HBV and HCV Therapy. Viruses 2009, 1(3), 484-509. doi:10.3390/v1030484

Related:

• Hepatitis B and C viruses
• Hepatitis C Virus: a mountain to climb
• Does the outcome of HCV infection vary with the infecting virus type?
• Plugging the holes in hepatitis C virus antiviral therapy