Epidemics of jaundice were reported as far back as ancient Greek and Chinese history. In past centuries, outbreaks of hepatitis were especially observed in association with wars and military conflict. In the second half of the 20th century, it became clear that there were several etiologically and epidemiologically distinct forms of hepatitis. The form transmitted primarily by fecal-oral contamination, which has a short incubation period and usually occurs as an epidemic, is caused by the hepatitis A virus (HAV), whereas the causal agents of the parenterally transmitted disease with a long incubation phase are caused by the hepatitis B and C viruses (HBV and HCV).
HAV is the major viral agent responsible for acute hepatitis. In the United States, there were approximately 19,000 acute clinical cases in 2005 [1]. Infections with HBV and HCV were responsible for 15,000 and 3200 cases, respectively. Since 1992, an inactivated hepatitis A vaccine has been available and has significantly lowered the number of cases in developed countries where the disease is sporadic or occasionally epidemic. In underdeveloped countries, HAV is endemic, with vast swathes of the population becoming infected early in life and acquiring lifelong immunity. Increasing standards of living have led to a decrease in the incidence of acute hepatitis A. Primary infection is often delayed, occurring later in life. As a consequence, the number of people susceptible to infection and the risk of suffering from clinically evident symptoms has significantly increased in developing countries. Due to its high cost, the existing vaccine is primarily used in the developed world and with individuals at high risk, including travelers to endemic areas as well as children and employees in closed communities (e.g. daycare centers). A live vaccine based on an attenuated viral variant was recently developed in China [2].
The successful propagation of HAV in cultured primate cells constituted a major breakthrough in detailed molecular studies and vaccine development [3,4]. Once sufficient amounts of purified virus became available, its RNA genome was reverse-transcribed and a HAV cDNA copy became available for molecular cloning [5], thus opening up means to obtaining a detailed characterization of this atypical and challenging picornavirus.
VIRAL GENES AND PROTEINS
HAV is the sole member of the genus Hepatovirus in the Picornaviridae family, which includes such important human and animal pathogens as poliovirus, rhinovirus, and aphthovirus, the agent responsible for foot-and-mouth disease. Poliovirus is the prototypical member of this family; as such, it was investigated intensively and the understanding of the molecular biology of HAV greatly benefited from these studies. However, in many molecular and biological aspects HAV is clearly different from other picornaviruses, making this virus a hot topic in molecular virology. Its uniquely protracted and persistent replication in cell culture is exceptional, and has complicated its molecular study. In general, analysis of the HAV genome structure and the functions of its proteins have relied on recombinant clones expressed in Escherichia coli and mammalian cells.
The HAV genome is a linear RNA molecule of messenger-sense polarity and 7500 base-lengths. A small polypeptide (VPg = 3B) is covalently linked to the 5´end of the RNA (see Figure 1 for the location of genetic elements and proteins on the viral genome). Like all picornaviral genomes, the HAV genome contains 3 parts with (i) a 5´non-translated region (5´NTR) of about 740 bases; (ii) a large open reading frame encoding a single polyprotein with a molecular mass of approximately 250 kDa; and (iii) a short 3´NTR with a poly(A) tail. The 5´NTR can be divided into 2 functionally distinct domains that both fold into stable secondary and tertiary RNA structures and that play essential roles in viral genome replication and translation. Whereas the terminal structure in the HAV 5´NTR is required for RNA synthesis, the following 500-nucleotide residues fold into a highly structured entity that is termed internal ribosomal entry site (IRES) [6]. The HAV IRES allows cap-independent translation initiation with the aid of host proteins that differ substantially from other picornaviruses [7]. Similar to the 5´NTR and possibly in synergy with it, the 3´NTR has functions in RNA and protein synthesis. In addition to the terminal cis-acting RNA elements, an intragenomic RNA stem-loop structure was recently identified for various members of the Picornaviridae family. This cis-acting replication element (CRE) has so far not been identified in the HAV genome. Overall, their specific binding to viral and host proteins mediates the function of these terminal and intragenomic RNA structures in the time and space regulation of the divergent processes for which the viral RNA genome acts as the template, namely viral translation and replication.
 | Figure 1. Schematic representation of the HAV genome and polyprotein processing of the structural proteins. The upper line (A) represents the viral positive sense RNA genome 7500 nucleotides in length with the large open reading frame (ORF) shown as a hatched block. At its 5´end, the genome is linked to the viral protein 3B (= VPg). The first 5´terminal 736 nucleotides are nontranslated. They include cis-acting RNA structures involved in genome replication and translation. At the 3´end, the non-translated region (NTR) functions as a cis-acting replication element. The 3´poly(A) tail of approximately 60 residues is essential for RNA stability and efficient translation.
The bottom section of the figure (B) depicts the proteolytic cascade yielding the structural proteins. The primary translation product is the polyprotein shown with the position and names of the 11 mature viral proteins. The primary cleavage in the polyprotein (the site is indicated by ▼ ) results in the production of the precursors of the structural (P1-2A) and non-structural proteins (P2-P3). Secondary cleavages within P1-2A occur in a coordinated manner and lead to the mature proteins that assemble to form the viral capsid. The mechanisms or enzymes involved in the 2 maturation cleavages (VP0 yielding VP4 and VP2; removal of 2A from VP1-2A) are as yet unknown. Cleavage of the precursor of the non-structural proteins most likely occurs on vesicle membranes and results in the formation of the replication complex. The cleavage cascade yielding intermediate and mature non-structural proteins is not depicted. |
The central region of the viral RNA encodes a single polyprotein with domains P1-2A, P2, and P3. Whereas domain P1-2A contains the capsid proteins VP1 (or VP1-2A), VP2, VP3, and VP4, all polypeptides encoded in domains P2 and P3 appear to be components of the viral replication complex (RC) that provides the structure for and catalyzes viral genome replication. As part of the viral polyprotein itself, only the virus-encoded proteinase 3C and its precursor polypeptides 3ABC, 3BC and 3CD catalyze almost all cleavages within the polyprotein and are therefore essential players in viral protein formation [8,9].
Time-regulated proteolytic release of viral proteins from the primary translation product is the key step in the gene expression strategy of many positive strand RNA viruses, been directed towards understanding the regulatory role of the viral proteinase. The bottom section of Figure 1 shows the proteolytic processing cascade producing intermediate and mature viral proteins. Intermediate and mature processing products have diverse functions, thus extending the limited coding capacity of the viral genome. The proposed or known biochemical/biophysical properties and functions of these cleavage products are summarized in Table 1.
 | TABLE 1. Biochemical and biophysical properties of HAV non-structural proteins |
HAV proteins 3C and 2A deserve special mention: 3C has been examined in great detail and 2A is unique in the Picornaviridae family. HAV 3C was the first picornaviral proteinase to have its crystal structure described [10,11]. The RNA-binding capacity renders the proteolytically active polypeptides 3C, 3ABC, 3BC and 3CD multifunctional and important in viral RNA synthesis [12]. The role of 3C to affect cell metabolism by specifically cleaving host proteins is the focus of ongoing studies. In contrast to the analog protein in other picornaviral genera, HAV protein 2A has no proteolytic function. So far, HAV 2A has only been found fused to the structural protein VP1; as well, its function as a signal for capsid assembly is unique [13-17]. Based on these findings, HAV 2A has to be considered a domain in the major structural protein VP1. The molecular biology of HAV has been reviewed in detail elsewhere [18,19].
VIRAL REPLICATION
In the first step of the viral life cycle (see Figure 2), HAV binds to its cell surface receptor, known as HAVcr [20]. HAVcr is a mucin-like integral membrane glycoprotein whose exact role in viral entry is not clear. This protein is not selectively expressed in the liver and hence liver tropism does not appear to be defined by the cell receptor. Interestingly, an alternate binding and entry mechanism mediated by IgA molecules has been proposed for HAV [21]. Upon binding and probably receptor-mediated endocytosis, it is assumed that the HAV particle structure is modified in such a way that the RNA genome is uncoated and released into the cytoplasm [22]. The incoming viral RNA genome initially serves as the template of translation that is cap-independent. The primary translation product, the polyprotein, is cleaved into structural proteins and non-structural proteins that presumably assemble into an RC together with host proteins. Located on virus-induced membrane vesicles, the RC catalyzes viral genome replication [23]. Viral RNA synthesis is template- and primer-dependent and proceeds through a negative strand intermediate. The subsequently formed positive RNA strands are either used as translation or replication templates or packaged by the structural proteins to form infectious viral capsids. HAV structural proteins are not essential for genome replication. Therefore, viral genome translation and replication have been studied using a viral replicon that encodes a reporter gene in the place of the structural gene [24,25]. Various antiviral intervention strategies (e.g. RNA interference, antiviral compounds) have been applied to improve our understanding of host and viral components in the viral life cycle [26,27]. Whereas most picornaviruses shut off host cell metabolism by cleaving proteins essential for host translation and transcription, HAV replicates persistently without inducing gross changes in cellular functions and morphology [28,29]. This remains an elusive subject of ongoing research: to elucidate the mechanism as well as identify which cell organelles viral particles use to exit the cell without causing lysis. The cell exit strategy appears to be tightly connected with capsid maturation and the removal of the assembly signal 2A by a host proteinase [15,17]. Interestingly, HAV release from polarized cells in culture occurs almost exclusively on the apical side [30]. This might mimic the directional secretion of the hepatocyte into the biliary system. Although studies on virus cell interactions have been initiated [31,32], the host cells´ innate antiviral response and their pro- and anti-apoptotic properties need further assessment.
 | Figure 2. The HAV life cycle.
HAV binds to the cellular receptor (1). The binding-induced conformational change of the viral particle allows RNA genome uncoating and release into the cytoplasm (2). The messenger-sense (+) RNA associates with ribosomes and translation is initiated. A polyprotein is generated that is proteolytically cleaved by the viral proteinase 3C into the structural proteins (SP) that assemble to form the viral capsid (C, 5). The non-structural proteins associate with host proteins to form the replication complex (RC) that catalyzes viral genome replication. Viral proteins 2B and 2C induce the formation of membrane vesicles that are the site of viral RNA synthesis. The (+) strand RNA is copied through a (-) strand intermediate (dashed line, 4a) into multiple new (+) strands (solid line, 4b). Some (+) strand RNA molecules are again translated, others are encapsidated by structural proteins to initially form immature particles (hatched hexagon, 6). Before or concomitant with maturation the virion exits the cell (7). |
THE HAV PARTICLE
The non-enveloped HAV has an icosahedral symmetry with a diameter of 27 to 30 nm. HAV is morphologically indistinguishable from other picornaviruses. The mature particle has a density of 1.34 g/mL and a sedimentation coefficient of 160S. Immature or empty particles are not as dense and sediment at a slower rate [33]. The HAV capsid exhibits high acid stability, a prerequisite for its passage through the digestive tract [34,35]. The viral capsid is composed of 12 pentamers, each one containing 5 copies of structural proteins VP1, VP2, VP3, and VP4. To date, the atomic structure of the HAV particle has not been determined and its distinct surface properties remain unknown. When viral RNA is packaged, capsid maturation can generate specific surface topology, which is required for the particle's ability to infect. This is also accompanied by an increase in physical stability. Maturation shifts the particle into a state where it can specifically and functionally interact with the cell receptor, allowing the subsequent uncoating of the viral genome. Maturation cleavage of VP0 into VP2 and VP4 occurs inside the viral particle and requires the presence of encapsidated RNA [36]. Unique among picornaviruses, a second maturation step (cleavage of VP1-2A) is catalyzed by an as yet unidentified host proteinase [15,17]. The involvement of a host proteinase in HAV maturation and possibly in the cell exit strategy is reminiscent of enveloped RNA viruses that require furin or other host proteinases to achieve full infectivity. It is tempting to speculate that this step might relate to liver tropism and thus be required for the directional and noncytolytic release of HAV from the infected hepatocyte.
GENETIC DIVERSITY AND ANTIGENICITY
In contrast to other picornaviruses, only one human HAV serotype exists. This specific feature of HAV is highly relevant to reinfection, serological diagnosis, and the efficiency of available vaccines that confer worldwide protection. The immunodominant neutralization site is discontinuous and depends on the particle's native conformation. For this reason, it is likely that a subunit hepatitis A vaccine will not be protective. In spite of the unique HAV serotype, some genetic diversity has been evident from the sequencing data obtained from a large number of HAV isolates. Based on sequence variations at the VP1/2A junction, genotypes and subgenotypes have been defined [37,38]. In addition to human isolates, a simian viral variant has also been identified [39].
Propagation in cell culture and host range
Owing to its slow replication mode, the isolation of wyldetype HAV in cell culture was unsuccessful for many years. Finally in 1978, various human and monkey cells were found to support HAV growth and several viral strains were adapted to cell culture [3,4]. Adaptation to growth in cell culture is reached by serial passages in cell cultures with a concomitant accumulation of mutations mainly in the 5´NTR and the P2 domain [40,41]. Viral replication is generally demonstrated by the detection of viral antigen by various immunological methods or of the viral genome by RT-PCR. In most HAV-infected cells, the induction of interferon has not been observed [31,42]. In spite of intensive genetic studies, the molecular basis for the slow and non-lytic replication in cell culture remains unknown. Interestingly, the protracted time course, low viral yields and the propensity to establish persistent infections in cell culture are specific features that HAV shares with other hepatitis viruses, i.e. HBV and HCV. This convergence of members of different families remains a fascinating conundrum for viral hepatologists.
Attempts to experimentally transmit HAV to various animals has indicated that susceptibility is mostly limited to primates [43,44]. Disease in marmosets, owl monkeys, and macaques resembles that in humans, but its course is usually milder. Among the various small animals tested, guinea pigs were able to support replication of a specifically adapted HAV strain [45]. Overall, our knowledge of HAV pathogenesis remains limited due to the lack of a reliable and convenient small animal model.
PATHOGENESIS AND PATHOLOGY
Detailed descriptions of the natural history and clinical features of HAV are given elsewhere [18,46,47], and in this monograph]. In brief, acute HAV infection is contracted orally. The source of infection is usually water or food contaminated with human fecal matter containing shed virus. Owing to its acid-resistance, HAV can survive in gastric pH. The precise mechanism of viral uptake in the gastrointestinal tract and passage to the liver is still unknown. Based on the finding that the IgA antibody-virus complex is taken up by liver cells, it was suggested that during its passage in the intestine, "HAV takes a detour to the liver on its way through the digestive tract thereby evading the immediate host defense" [48]. Viral replication either in intestinal epithelial cells or in Peyer's patches has been suggested, but not experimentally proven [49]. In the liver, the virus replicates in hepatocytes, causing subsequent degeneration. Infection is associated with a wide spectrum of clinical outcomes ranging from asymptomatic or subclinical illness with altered liver enzyme profiles, to classical hepatitis with jaundice, and even fulminant hepatic failure. The most important factor responsible for the different disease patterns is the age of the infected person: clinical jaundice occurs in less than 5% of children 3 years of age and under, but in over 80% of adults. After an incubation period of 2 to 7 weeks, non-specific symptoms such as fever, malaise, nausea and flulike complaints arise. Dark urine is usually the first objective sign of hepatitis followed by pale, acholic feces and jaundice of the sclera and skin. Large amounts of HAV are produced in infected hepatocytes that secrete the virus into the intestinal tract via the biliary system. Activation of cells in the reticuloendothelial system of the sinusoids and the portal tract marks the onset of immune mechanisms. Fecal shedding of the virus peaks just before the onset of hepatic injury and occurs in parallel with the viremic phase. In most cases, the symptoms usually disappear after 2 or 3 weeks. Liver enzymes are normal after 4 weeks. The detection of large amounts of virus prior to the onset of clinical symptoms supports the notion that the action of HAVspecific cytotoxic T lymphocytes (CTL) rather than the direct cytopathic effect of HAV is the cause of liver cell destruction and finally viral eradication from the organism [50]. As a typical measure of an acute infection, the IgM response is detectable 3 to 10 weeks post-infection. IgG antibodies occur soon after IgM and persist for years, conferring lifelong immunity. HAV-specific IgA antibodies can be detected in serum, saliva, and stool. Their role in HAV transmission from the intestine to the liver has recently been proposed [21,48].
CONCLUSION
HAV is still a significant cause of morbidity and even mortality and therefore remains a hot topic for virologists. Great success has been achieved by preventive immunization with the killed hepatitis A vaccine, which provides long-lasting protection from infection by all known viral variants. The unique molecular and biological features of HAV are elusive and still present a challenge to molecular virologists. Besides an improved understanding of the infectious agent, studies on this hepatotropic picornavirus will help in further elucidating liver cell metabolism, in particular with regard to its antiviral mechanisms.
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