Virology and Pathogenesis of Hepatitis B

Introduction

Hepatitis B virus (HBV) has had a very long and successful relationship with humans. The detection of hepadnavirus genomes in Mesozoic birds and the estimation that HBV was already present in early humans at least 40,000 years ago support a coevolution scenario with humans. The overall success of HBV as a human virus is also displayed by these simple numbers: a third of the human population has been in contact with the virus, and approximately 200 million to 300 million people are chronically infected. This relationship is, however, not without consequences for humans because approximately 20% of people with active viral replication develop different grades of liver inflammation (chronic hepatitis) that can lead to cirrhosis or liver cancer. Understanding the fundamental steps of HBV biology and how the host's immunity interacts with HBV and how this interaction can lead to pathologic consequences or potential functional cure is the focus of this chapter.

Hepatitis B Virus: Host and Model Systems

HBV is a small, enveloped DNA virus that belongs to the family Hepadnaviridae . Various model systems ( Tables 31-1 and 31-2 ) have been used to characterize the HBV replication cycle, identify novel antiviral targets, study HBV pathogenesis, and assess the efficacy of novel therapeutic strategies. Human hepatocytes are the natural target cells of HBV. These cells can be isolated from liver resections and retain susceptibility to HBV infection for a short period in culture. However, the accessibility to fresh human liver resections and the quality and variability of the individual preparations limit their use. Primary hepatocytes of Tupaia belangeri are also susceptible to HBV infection but the difficulty in rearing these animals and the absence of Tupaia -specific reagents for functional studies limit their use. As an alternative to primary cell cultures, the human hepatoma Huh7 and HepG2 cell lines were used for many years to perform in vitro experiments on HBV. Those cells allow HBV replication and viral particle assembly but they are not susceptible to infection because of the lack of expression of the HBV receptor(s). Alternatively, HepaRG cells, which are liver progenitors, can be used for in vitro studies because they become susceptible to HBV infection after differentiation in culture. However, infection rates are low and spread of virus within the cultures was never observed. With the recent discovery of sodium-taurocholate cotransporting polypeptide (NTCP) as an HBV/hepatitis D virus receptor, HepG2 and Huh7 cell lines (over)expressing NTCP have been generated and are susceptible to HBV infection. However, their capacity to allow virus propagation remains to be determined as does their relevance for studies of virus-host cell interactions because of their transformed nature (see Table 31-1 ). Finally, a recent study showed that micropatterned cocultures of primary human hepatocytes or induced pluripotent stem cells and stromal cells differentiated into hepatocyte-like cells, with fibroblasts maintaining prolonged HBV infection.

Although cell culture models are very valuable to characterize defined aspects of the viral life cycle, in vivo models are necessary to study HBV pathogenesis and new antiviral strategies, including immunotherapies (see Table 31-2 ). HBV has an extremely narrow host range because it infects only hominoid apes, including chimpanzees. The latter have been used in pivotal studies deciphering host responses during acute HBV infection but are no longer available for experimental studies. Therefore, macaques, which have a 93% sequence identity with humans and are frequently used in toxicology, are being considered as an alternative model to study viral hepatitis. There are also various HBV-related viruses such as duck HBV and woodchuck hepatitis virus that have been invaluable models to study HBV infection (see Table 32-2 ). Mice are not naturally susceptible to HBV infection but they can be humanized to study HBV infection in vivo . Different models have been used to generate human liver chimeric (HuHEP) mice. These mice are characterized by both a progressive degeneration of mouse liver cells and immune deficiency, thereby allowing engraftment of human hepatocytes. Inoculation of HuHEP mice with HBV leads to productive infection, and these mice have been used for proof-of-concept studies assessing the efficacy of novel antiviral strategies. To allow assessment of viral pathogenesis in the context of a functional human immune system and to test immunotherapies, a double-humanized mouse, carrying both a humanized immune system and human hepatocytes was created and used for initial pathogenetic studies. The adherence of these mice to key aspects of chronic viral hepatitis in humans needs, however, to be fully evaluated. Finally, other immunocompetent mouse models of chronic HBV infection have been established by use of low doses of adenovirus, adeno-associated virus–mediated gene transfer, or hydrodynamic transfection of the HBV genome. Because HBV production is mediated by a different virus or forced through transfection, these mice do not recapitulate the physiologic steps of HBV infection but can be used to answer specific immunopathologic questions.

Hepatitis B Virus Biology

Hepatitis B Virus Structure, Genome, and Proteins

HBV particles, called Dane particles , are spherical lipid-containing structures with a diameter of approximately 42 nm ( Fig. 31-1 ). The inner shell of the virus consists of an icosahedral nucleocapsid, which is assembled from 120 dimers of the core protein. The nucleocapsid is covered with a membrane containing three forms of the viral envelope proteins—large (L), middle (M), and small (S)—that are acquired together with the host's lipids during budding into the endoplasmic reticulum. The three surface proteins are commonly defined as hepatitis B surface antigens. They are translated from their own start codons but share the same C-terminal amino acids, called the S domain . As a consequence, the M protein contains an extra domain called the pre-S2 domain , compared with the S protein, and the L protein contains two extra domains: pre-S2 and pre-S1. Nucleocapsids contain a single copy of the HBV genome consisting of a 3.2-kb partially double-stranded relaxed circular DNA (rcDNA) molecule. This rcDNA is covalently linked to the viral polymerase at the 5′ end of the complete strand, also called viral minus-strand DNA . Besides the Dane particles, HBV infection also leads to secretion of subviral particles, which consist of empty viral envelopes with filamentous or spherical shapes and empty virions, which contain both the outer envelope and the inner capsid shell but no viral genome. Subviral particles are produced in huge excess (100- to 100,000-fold over Dane particles) into the blood. They are thought to facilitate virus spread and persistence in the host by adsorbing virus-neutralizing antibodies. The HBV polymerase is a multifunctional protein that plays a central role in viral replication. It is divided into four separate domains, from the N-terminus: the TP domain, which is essential for the priming of the reverse transcription; the RT domain, which harbors the polymerase active site essential for DNA polymerization; the RNase H domain, which is responsible for degrading the pregenomic RNA (pgRNA) template during minus-strand DNA synthesis; and the spacer region, which is the least conserved of the four domains and is dispensable for all known functions of the enzyme. In addition to polymerase and the structural proteins, the HBV genome also encodes three nonstructural proteins. Secreted hepatitis B e antigens (HBeAgs) have been suggested to have immunoregulatory functions, whereas the X protein (HBx) is at least essential to initiate and maintain HBV transcription (see Fig. 31-1 ). In addition, hepatitis B spliced protein expression is the result of HBV messenger RNA splicing, and its role in the virus-induced pathogenesis remains to be fully demonstrated.