HAV is a nonenveloped 27-nm, heat-, acid-, and ether-resistant RNA virus in the Hepatovirus genus of the picornavirus family (Fig. 360-1). Its virion contains four capsid polypeptides, designated VP1 to VP4, which are cleaved posttranslationally from the polyprotein product of a 7500-nucleotide genome. Inactivation of viral activity can be achieved by boiling for 1 min, by contact with formaldehyde and chlorine, or by ultraviolet irradiation. Despite nucleotide sequence variation of up to 20% among isolates of HAV, and despite the recognition of four genotypes affecting humans, all strains of this virus are immunologically indistinguishable and belong to one serotype. Hepatitis A has an incubation period of ~4 weeks. Its replication is limited to the liver, but the virus is present in the liver, bile, stools, and blood during the late incubation period and acute preicteric/presymptomatic phase of illness. Despite slightly longer persistence of virus in the liver, fecal shedding, viremia, and infectivity diminish rapidly once jaundice becomes apparent. HAV can be cultivated reproducibly in vitro.
Electron micrographs of hepatitis A virus particles and serum from a patient with hepatitis B. Left: 27-nm hepatitis A virus particles purified from stool of a patient with acute hepatitis A and aggregated by antibody to hepatitis A virus. Right: Concentrated serum from a patient with hepatitis B, demonstrating the 42-nm virions, tubular forms, and spherical 22-nm particles of hepatitis B surface antigen. 132,000×. (Hepatitis D resembles 42-nm virions of hepatitis B but is smaller, 35–37 nm; hepatitis E resembles hepatitis A virus but is slightly larger, 32–34 nm; hepatitis C has been visualized as a 55-nm particle.)
Antibodies to HAV (anti-HAV) can be detected during acute illness when serum aminotransferase activity is elevated and fecal HAV shedding is still occurring. This early antibody response is predominantly of the IgM class and persists for several (~3) months, rarely for 6–12 months. During convalescence, however, anti-HAV of the IgG class becomes the predominant antibody (Fig. 360-2). Therefore, the diagnosis of hepatitis A is made during acute illness by demonstrating anti-HAV of the IgM class. After acute illness, anti-HAV of the IgG class remains detectable indefinitely, and patients with serum anti-HAV are immune to reinfection. Neutralizing antibody activity parallels the appearance of anti-HAV, and the IgG anti-HAV present in immune globulin accounts for the protection it affords against HAV infection.
Scheme of typical clinical and laboratory features of hepatitis A virus (HAV). ALT, alanine aminotransferase.
HBV is a DNA virus with a remarkably compact genomic structure; despite its small, circular, 3200-bp size, HBV DNA codes for four sets of viral products with a complex, multiparticle structure. HBV achieves its genomic economy by relying on an efficient strategy of encoding proteins from four overlapping genes: S, C, P, and X (Fig. 360-3), as detailed below. Once thought to be unique among viruses, HBV is now recognized as one of a family of animal viruses, hepadnaviruses (hepatotropic DNA viruses), and is classified as hepadnavirus type 1. Similar viruses infect certain species of woodchucks, ground and tree squirrels, and Pekin ducks, to mention the most carefully characterized. Like HBV, all have the same distinctive three morphologic forms, have counterparts to the envelope and nucleocapsid virus antigens of HBV, replicate in the liver but exist in extrahepatic sites, contain their own endogenous DNA polymerase, have partially double-strand and partially single-strand genomes, are associated with acute and chronic hepatitis and hepatocellular carcinoma, and rely on a replicative strategy unique among DNA viruses but typical of retroviruses. Instead of DNA replication directly from a DNA template, hepadnaviruses rely on reverse transcription (effected by the DNA polymerase) of minus-strand DNA from a “pregenomic” RNA intermediate. Then plus-strand DNA is transcribed from the minus-strand DNA template by the DNA-dependent DNA polymerase and converted in the hepatocyte nucleus to a covalently closed circular DNA, which serves as a template for messenger RNA and pregenomic RNA. Viral proteins are translated by the messenger RNA, and the proteins and genome are packaged into virions and secreted from the hepatocyte. Although HBV is difficult to cultivate in vitro in the conventional sense from clinical material, several cell lines have been transfected with HBV DNA. Such transfected cells support in vitro replication of the intact virus and its component proteins.
Compact genomic structure of hepatitis B virus (HBV). This structure, with overlapping genes, permits HBV to code for multiple proteins. The S gene codes for the “major” envelope protein, HBsAg. Pre-S1 and pre-S2, upstream of S, combine with S to code for two larger proteins, “middle” protein, the product of pre-S2 + S, and “large” protein, the product of pre-S1 + pre-S2 + S. The largest gene, P, codes for DNA polymerase. The C gene codes for two nucleocapsid proteins, HBeAg, a soluble, secreted protein (initiation from the pre-C region of the gene), and HBcAg, the intracellular core protein (initiation after pre-C). The X gene codes for HBxAg, which can transactivate the transcription of cellular and viral genes; its clinical relevance is not known, but it may contribute to carcinogenesis by binding to p53.
VIRAL PROTEINS AND PARTICLES
Of the three particulate forms of HBV (Table 360-1), the most numerous are the 22-nm particles, which appear as spherical or long filamentous forms; these are antigenically indistinguishable from the outer surface or envelope protein of HBV and are thought to represent excess viral envelope protein. Outnumbered in serum by a factor of 100 or 1000 to 1 compared with the spheres and tubules are large, 42-nm, double-shelled spherical particles, which represent the intact hepatitis B virion (Fig. 360-1). The envelope protein expressed on the outer surface of the virion and on the smaller spherical and tubular structures is referred to as hepatitis B surface antigen (HBsAg). The concentration of HBsAg and virus particles in the blood may reach 500 μg/mL and 10 trillion particles per milliliter, respectively. The envelope protein, HBsAg, is the product of the S gene of HBV.
Envelope HBsAg subdeterminants include a common group-reactive antigen, a, shared by all HBsAg isolates and one of several subtype-specific antigens—d or y, w or r —as well as other specificities. Hepatitis B isolates fall into one of at least eight subtypes and ten genotypes (A–J). Geographic distribution of genotypes and subtypes varies; genotypes A (corresponding to subtype adw) and D (ayw) predominate in the United States and Europe, whereas genotypes B (adw) and C (adr) predominate in Asia. Clinical course and outcome are independent of subtype, but genotype B appears to be associated with less rapidly progressive liver disease and cirrhosis and a lower likelihood, or delayed appearance, of hepatocellular carcinoma than genotype C or D. Patients with genotype A are more likely to clear circulating viremia and to achieve HBeAg and HBsAg seroconversion, both spontaneously and in response to antiviral therapy. In addition, “precore” mutations are favored by certain genotypes (see below).
TABLE 360-1Nomenclature and Features of Hepatitis Viruses ||Download (.pdf) TABLE 360-1 Nomenclature and Features of Hepatitis Viruses
|Hepatitis Type ||Virus Particle, nm ||Morphology ||Genomea ||Classification ||Antigen(s) ||Antibodies ||Remarks |
|HAV ||27 ||Icosahedral nonenveloped ||7.5-kb RNA, linear, ss, + ||Hepatovirus ||HAV ||Anti-HAV ||Early fecal shedding Diagnosis: IgM anti-HAV Previous infection: IgG anti-HAV |
|HBV || |
Double-shelled virion (surface and core) spherical
Spherical and filamentous; represents excess virus coat material
|3.2-kb DNA, circular, ss/ds ||Hepadnavirus || |
Bloodborne virus; carrier state
Acute diagnosis: HBsAg, IgM anti-HBc
Chronic diagnosis: IgG anti-HBc, HBsAg
Markers of replication: HBeAg, HBV DNA
Liver, lymphocytes, other organs
Nucleocapsid contains DNA and DNA polymerase; present in hepatocyte nucleus; HBcAg does not circulate; HBeAg (soluble, nonparticulate) and HBV DNA circulate—correlate with infectivity and complete virions
HBsAg detectable in >95% of patients with acute hepatitis B; found in serum, body fluids, hepatocyte cytoplasm; anti-HBs appears following infection—protective antibody
|HCV ||Approx. 50–80 ||Enveloped ||9.4-kb RNA, linear, ss, + ||Hepacivirus || |
|Anti-HCV || |
Bloodborne agent, formerly labeled non-A, non-B hepatitis
Acute diagnosis: anti-HCV (C33c, C22-3, NS5), HCV RNA
Chronic diagnosis: anti-HCV (C100-3, C33c, C22-3, NS5) and HCV RNA; cytoplasmic location in hepatocytes
|HDV ||35–37 ||Enveloped hybrid particle with HBsAg coat and HDV core ||1.7-kb RNA, circular, ss, – ||Resembles viroids and plant satellite viruses (genus Deltavirus) || |
Defective RNA virus, requires helper function of HBV (hepadnaviruses); HDV antigen (HDAg) present in hepatocyte nucleus
Diagnosis: anti-HDV, HDV RNA; HBV/HDV co-infection—IgM anti-HBc and anti-HDV; HDV superinfection—IgG anti-HBc and anti-HDV
|HEV ||32–34 ||Nonenveloped icosahedral ||7.6-kb RNA, linear, ss, + ||Hepevirus ||HEV antigen ||Anti-HEV || |
Agent of enterically transmitted hepatitis; rare in United States; occurs in Asia, Mediterranean countries, Central America
Diagnosis: IgM/IgG anti-HEV (assays not routinely available); virus in stool, bile, hepatocyte cytoplasm
Upstream of the S gene are the pre-S genes (Fig. 360-3), which code for pre-S gene products, including receptors on the HBV surface for polymerized human serum albumin and for hepatocyte membrane proteins. The pre-S region actually consists of both pre-S1 and pre-S2. Depending on where translation is initiated, three potential HBsAg gene products are synthesized. The protein product of the S gene is HBsAg (major protein), the product of the S region plus the adjacent pre-S2 region is the middle protein, and the product of the pre-S1 plus pre-S2 plus S regions is the large protein. Compared with the smaller spherical and tubular particles of HBV, complete 42-nm virions are enriched in the large protein. Both pre-S proteins and their respective antibodies can be detected during HBV infection, and the period of pre-S antigenemia appears to coincide with other markers of virus replication, as detailed below; however, pre-S proteins have little clinical relevance and are not included in routine serologic testing repertoires.
The intact 42-nm virion contains a 27-nm nucleocapsid core particle. Nucleocapsid proteins are coded for by the C gene. The antigen expressed on the surface of the nucleocapsid core is hepatitis B core antigen (HBcAg), and its corresponding antibody is anti-HBc. A third HBV antigen is hepatitis B e antigen (HBeAg), a soluble, nonparticulate, nucleocapsid protein that is immunologically distinct from intact HBcAg but is a product of the same C gene. The C gene has two initiation codons, a precore and a core region (Fig. 360-3). If translation is initiated at the precore region, the protein product is HBeAg, which has a signal peptide that binds it to the smooth endoplasmic reticulum, the secretory apparatus of the cell, leading to its secretion into the circulation. If translation begins at the core region, HBcAg is the protein product; it has no signal peptide, it is not secreted, but it assembles into nucleocapsid particles, which bind to and incorporate RNA, and which, ultimately, contain HBV DNA. Also packaged within the nucleocapsid core is a DNA polymerase, which directs replication and repair of HBV DNA. When packaging within viral proteins is complete, synthesis of the incomplete plus strand stops; this accounts for the single-strand gap and for differences in the size of the gap. HBcAg particles remain in the hepatocyte, where they are readily detectable by immunohistochemical staining and are exported after encapsidation by an envelope of HBsAg. Therefore, naked core particles do not circulate in the serum. The secreted nucleocapsid protein, HBeAg, provides a convenient, readily detectable, qualitative marker of HBV replication and relative infectivity.
HBsAg-positive serum containing HBeAg is more likely to be highly infectious and to be associated with the presence of hepatitis B virions (and detectable HBV DNA, see below) than HBeAg-negative or anti-HBe-positive serum. For example, HBsAg-positive mothers who are HBeAg-positive almost invariably (>90%) transmit hepatitis B infection to their offspring, whereas HBsAg-positive mothers with anti-HBe rarely (10–15%) infect their offspring.
Early during the course of acute hepatitis B, HBeAg appears transiently; its disappearance may be a harbinger of clinical improvement and resolution of infection. Persistence of HBeAg in serum beyond the first 3 months of acute infection may be predictive of the development of chronic infection, and the presence of HBeAg during chronic hepatitis B tends to be associated with ongoing viral replication, infectivity, and inflammatory liver injury (except during the early decades after perinatally acquired HBV infection; see below).
The third and largest of the HBV genes, the P gene (Fig. 360-3), codes for HBV DNA polymerase; as noted above, this enzyme has both DNA-dependent DNA polymerase and RNA-dependent reverse transcriptase activities. The fourth gene, X, codes for a small, nonparticulate protein, hepatitis B x antigen (HBxAg), that is capable of transactivating the transcription of both viral and cellular genes(Fig. 360-3). In the cytoplasm, HBxAg effects calcium release (possibly from mitochondria), which activates signal-transduction pathways that lead to stimulation of HBV reverse transcription and HBV DNA replication. Such transactivation may enhance the replication of HBV, leading to the clinical association observed between the expression of HBxAg and antibodies to it in patients with severe chronic hepatitis and hepatocellular carcinoma. The transactivating activity can enhance the transcription and replication of other viruses besides HBV, such as HIV. Cellular processes transactivated by X include the human interferon γ gene and class I major histocompatibility genes; potentially, these effects could contribute to enhanced susceptibility of HBV-infected hepatocytes to cytolytic T cells. The expression of X can also induce programmed cell death (apoptosis). The clinical relevance of HBxAg is limited, however, and testing for it is not part of routine clinical practice.
SEROLOGIC AND VIROLOGIC MARKERS
After a person is infected with HBV, the first virologic marker detectable in serum within 1–12 weeks, usually between 8 and 12 weeks, is HBsAg (Fig. 360-4). Circulating HBsAg precedes elevations of serum aminotransferase activity and clinical symptoms by 2–6 weeks and remains detectable during the entire icteric or symptomatic phase of acute hepatitis B and beyond. In typical cases, HBsAg becomes undetectable 1–2 months after the onset of jaundice and rarely persists beyond 6 months. After HBsAg disappears, antibody to HBsAg (anti-HBs) becomes detectable in serum and remains detectable indefinitely thereafter. Because HBcAg is intracellular and, when in the serum, sequestered within an HBsAg coat, naked core particles do not circulate in serum, and therefore, HBcAg is not detectable routinely in the serum of patients with HBV infection. By contrast, anti-HBc is readily demonstrable in serum, beginning within the first 1–2 weeks after the appearance of HBsAg and preceding detectable levels of anti-HBs by weeks to months. Because variability exists in the time of appearance of anti-HBs after HBV infection, occasionally a gap of several weeks or longer may separate the disappearance of HBsAg and the appearance of anti-HBs. During this “gap” or “window” period, anti-HBc may represent the only serologic evidence of current or recent HBV infection, and blood containing anti-HBc in the absence of HBsAg and anti-HBs has been implicated in transfusion-associated hepatitis B. In part because the sensitivity of immunoassays for HBsAg and anti-HBs has increased, however, this window period is rarely encountered. In some persons, years after HBV infection, anti-HBc may persist in the circulation longer than anti-HBs. Therefore, isolated anti-HBc does not necessarily indicate active virus replication; most instances of isolated anti-HBc represent hepatitis B infection in the remote past. Rarely, however, isolated anti-HBc represents low-level hepatitis B viremia, with HBsAg below the detection threshold, and, occasionally, isolated anti-HBc represents a cross-reacting or false-positive immunologic specificity. Recent and remote HBV infections can be distinguished by determination of the immunoglobulin class of anti-HBc. Anti-HBc of the IgM class (IgM anti-HBc) predominates during the first 6 months after acute infection, whereas IgG anti-HBc is the predominant class of anti-HBc beyond 6 months. Therefore, patients with current or recent acute hepatitis B, including those in the anti-HBc window, have IgM anti-HBc in their serum. In patients who have recovered from hepatitis B in the remote past as well as those with chronic HBV infection, anti-HBc is predominantly of the IgG class. Infrequently, in ≤1–5% of patients with acute HBV infection, levels of HBsAg are too low to be detected; in such cases, the presence of IgM anti-HBc establishes the diagnosis of acute hepatitis B. When isolated anti-HBc occurs in the rare patient with chronic hepatitis B whose HBsAg level is below the sensitivity threshold of contemporary immunoassays (a low-level carrier), anti-HBc is of the IgG class. Generally, in persons who have recovered from hepatitis B, anti-HBs and anti-HBc persist indefinitely.
Scheme of typical clinical and laboratory features of acute hepatitis B. ALT, alanine aminotransferase.
The temporal association between the appearance of anti-HBs and resolution of HBV infection as well as the observation that persons with anti-HBs in serum are protected against reinfection with HBV suggests that anti-HBs is the protective antibody. Therefore, strategies for prevention of HBV infection are based on providing susceptible persons with circulating anti-HBs (see below). Occasionally, in ~10% of patients with chronic hepatitis B, low-level, low-affinity anti-HBs can be detected. This antibody is directed against a subtype determinant different from that represented by the patient’s HBsAg; its presence is thought to reflect the stimulation of a related clone of antibody-forming cells, but it has no clinical relevance and does not signal imminent clearance of hepatitis B. These patients with HBsAg and such nonneutralizing anti-HBs should be categorized as having chronic HBV infection.
The other readily detectable serologic marker of HBV infection, HBeAg, appears concurrently with or shortly after HBsAg. Its appearance coincides temporally with high levels of virus replication and reflects the presence of circulating intact virions and detectable HBV DNA (with the notable exception of patients with precore mutations who cannot synthesize HBeAg—see “Molecular Variants”). Pre-S1 and pre-S2 proteins are also expressed during periods of peak replication, but assays for these gene products are not routinely available. In self-limited HBV infections, HBeAg becomes undetectable shortly after peak elevations in aminotransferase activity, before the disappearance of HBsAg, and anti-HBe then becomes detectable, coinciding with a period of relatively lower infectivity (Fig. 360-4). Because markers of HBV replication appear transiently during acute infection, testing for such markers is of little clinical utility in typical cases of acute HBV infection. In contrast, markers of HBV replication provide valuable information in patients with protracted infections.
Departing from the pattern typical of acute HBV infections, in chronic HBV infection, HBsAg remains detectable beyond 6 months, anti-HBc is primarily of the IgG class, and anti-HBs is either undetectable or detectable at low levels (see “Laboratory Features”) (Fig. 360-5). During early chronic HBV infection, HBV DNA can be detected both in serum and in hepatocyte nuclei, where it is present in free or episomal form. This relatively highly replicative stage of HBV infection is the time of maximal infectivity and liver injury; HBeAg is a qualitative marker and HBV DNA a quantitative marker of this replicative phase, during which all three forms of HBV circulate, including intact virions. Over time, the relatively replicative phase of chronic HBV infection gives way to a relatively nonreplicative phase. This occurs at a rate of ~10% per year and is accompanied by seroconversion from HBeAg to anti-HBe. In many cases, this seroconversion coincides with a transient, usually mild, acute hepatitis-like elevation in aminotransferase activity, believed to reflect cell-mediated immune clearance of virus-infected hepatocytes. In the nonreplicative phase of chronic infection, when HBV DNA is demonstrable in hepatocyte nuclei, it tends to be integrated into the host genome. In this phase, only spherical and tubular forms of HBV, not intact virions, circulate, and liver injury tends to subside. Most such patients would be characterized as inactive HBV carriers. In reality, the designations replicative and nonreplicative are only relative; even in the so-called nonreplicative phase, HBV replication can be detected at levels of approximately ≤103 virions with highly sensitive amplification probes such as the polymerase chain reaction (PCR); below this replication threshold, liver injury and infectivity of HBV are limited to negligible. Still, the distinctions are pathophysiologically and clinically meaningful. Occasionally, nonreplicative HBV infection converts back to replicative infection. Such spontaneous reactivations are accompanied by reexpression of HBeAg and HBV DNA, and sometimes of IgM anti-HBc, as well as by exacerbations of liver injury. Because high-titer IgM anti-HBc can reappear during acute exacerbations of chronic hepatitis B, relying on IgM anti-HBc versus IgG anti-HBc to distinguish between acute and chronic hepatitis B infection, respectively, may not always be reliable; in such cases, patient history is invaluable in helping to distinguish de novo acute hepatitis B infection from acute exacerbation of chronic hepatitis B infection.
Scheme of typical laboratory features of wild-type chronic hepatitis B. HBeAg and hepatitis B virus (HBV) DNA can be detected in serum during the relatively replicative phase of chronic infection, which is associated with infectivity and liver injury. Seroconversion from the replicative phase to the relatively nonreplicative phase occurs at a rate of ~10% per year and is heralded by an acute hepatitis–like elevation of alanine aminotransferase (ALT) activity; during the nonreplicative phase, infectivity and liver injury are limited. In HBeAg-negative chronic hepatitis B associated with mutations in the precore region of the HBV genome, replicative chronic hepatitis B occurs in the absence of HBeAg.
Variation occurs throughout the HBV genome, and clinical isolates of HBV that do not express typical viral proteins have been attributed to mutations in individual or even multiple gene locations. For example, variants have been described that lack nucleocapsid proteins (commonly), envelope proteins (very rarely), or both. Two categories of naturally occurring HBV variants have attracted the most attention. One of these was identified initially in Mediterranean countries among patients with severe chronic HBV infection and detectable HBV DNA but with anti-HBe instead of HBeAg. These patients were found to be infected with an HBV mutant that contained an alteration in the precore region rendering the virus incapable of encoding HBeAg. Although several potential mutation sites exist in the pre-C region, the region of the C gene necessary for the expression of HBeAg (see “Virology and Etiology”), the most commonly encountered in such patients is a single base substitution, from G to A in the second to last codon of the pre-C gene at nucleotide 1896. This substitution results in the replacement of the TGG tryptophan codon by a stop codon (TAG), which prevents the translation of HBeAg. Another mutation, in the core-promoter region, prevents transcription of the coding region for HBeAg and yields an HBeAg-negative phenotype. Patients with such mutations in the precore region and who are unable to secrete HBeAg may have severe liver disease that progresses more rapidly to cirrhosis, or alternatively, they are identified clinically later in the course of the natural history of chronic hepatitis B, when the disease is more advanced. Both “wild-type” HBV and precore-mutant HBV can coexist in the same patient, or mutant HBV may arise late during wild-type HBV infection. In addition, clusters of fulminant hepatitis B in Israel and Japan were attributed to common-source infection with a precore mutant. Fulminant hepatitis B in North America and western Europe, however, occurs in patients infected with wild-type HBV, in the absence of precore mutants, and both precore mutants and other mutations throughout the HBV genome occur commonly, even in patients with typical, self-limited, milder forms of HBV infection. HBeAg-negative chronic hepatitis with mutations in the precore region is now the most frequently encountered form of hepatitis B in Mediterranean countries and in Europe. In the United States, where HBV genotype A (less prone to G1896A mutation) is prevalent, precore-mutant HBV is much less common; however, as a result of immigration from Asia and Europe, the proportion of HBeAg-negative hepatitis B–infected individuals has increased in the United States, and they now represent approximately 30–40% of patients with chronic hepatitis B. Characteristic of such HBeAg-negative chronic hepatitis B are lower levels of HBV DNA (usually ≤105 IU/mL) and one of several patterns of aminotransferase activity—persistent elevations, periodic fluctuations above the normal range, and periodic fluctuations between the normal and elevated range.
The second important category of HBV mutants consists of escape mutants, in which a single amino acid substitution, from glycine to arginine, occurs at position 145 of the immunodominant a determinant common to all HBsAg subtypes. This HBsAg alteration leads to a critical conformational change that results in a loss of neutralizing activity by anti-HBs. This specific HBV/a mutant has been observed in two situations, active and passive immunization, in which humoral immunologic pressure may favor evolutionary change (“escape”) in the virus—in a small number of hepatitis B vaccine recipients who acquired HBV infection despite the prior appearance of neutralizing anti-HBs and in HBV-infected liver transplant recipients treated with a high-potency human monoclonal anti-HBs preparation. Although such mutants have not been recognized frequently, their existence raises a concern that may complicate vaccination strategies and serologic diagnosis.
Different types of mutations emerge during antiviral therapy of chronic hepatitis B with nucleoside analogues; such “YMDD” and similar mutations in the polymerase motif of HBV are described in Chap. 362.
Hepatitis B antigens and HBV DNA have been identified in extrahepatic sites, including lymph nodes, bone marrow, circulating lymphocytes, spleen, and pancreas. Although the virus does not appear to be associated with tissue injury in any of these extrahepatic sites, its presence in these “remote” reservoirs has been invoked (but is not necessary) to explain the recurrence of HBV infection after orthotopic liver transplantation. The clinical relevance of such extrahepatic HBV is limited.
The delta hepatitis agent, or HDV, the only member of the genus Deltavirus, is a defective RNA virus that co-infects with and requires the helper function of HBV (or other hepadnaviruses) for its replication and expression. Slightly smaller than HBV, HDV is a formalin-sensitive, 35- to 37-nm virus with a hybrid structure. Its nucleocapsid expresses HDV antigen (HDAg), which bears no antigenic homology with any of the HBV antigens, and contains the virus genome. The HDV core is “encapsidated” by an outer envelope of HBsAg, indistinguishable from that of HBV except in its relative compositions of major, middle, and large HBsAg component proteins. The genome is a small, 1700-nucleotide, circular, single-strand RNA of negative polarity that is nonhomologous with HBV DNA (except for a small area of the polymerase gene) but that has features and the rolling circle model of replication common to genomes of plant satellite viruses or viroids. HDV RNA contains many areas of internal complementarity; therefore, it can fold on itself by internal base pairing to form an unusual, very stable, rodlike structure that contains a very stable, self-cleaving and self-ligating ribozyme. HDV RNA requires host RNA polymerase II for its replication in the hepatocyte nucleus via RNA-directed RNA synthesis by transcription of genomic RNA to a complementary antigenomic (plus strand) RNA; the antigenomic RNA, in turn, serves as a template for subsequent genomic RNA synthesis effected by host RNA polymerase I. HDV RNA has only one open reading frame, and HDAg, a product of the antigenomic strand, is the only known HDV protein; HDAg exists in two forms: a small, 195-amino-acid species, which plays a role in facilitating HDV RNA replication, and a large, 214-amino-acid species, which appears to suppress replication but is required for assembly of the antigen into virions. HDV antigens have been shown to bind directly to RNA polymerase II, resulting in stimulation of transcription. Although complete hepatitis D virions and liver injury require the cooperative helper function of HBV, intracellular replication of HDV RNA can occur without HBV. Genomic heterogeneity among HDV isolates has been described; however, pathophysiologic and clinical consequences of this genetic diversity have not been recognized. The clinical spectrum of hepatitis D is common to all eight genotypes identified, the predominant of which is genotype 1.
HDV can either infect a person simultaneously with HBV (co-infection) or superinfect a person already infected with HBV (superinfection); when HDV infection is transmitted from a donor with one HBsAg subtype to an HBsAg-positive recipient with a different subtype, HDV assumes the HBsAg subtype of the recipient, rather than the donor. Because HDV relies absolutely on HBV, the duration of HDV infection is determined by the duration of (and cannot outlast) HBV infection. HDV replication tends to suppress HBV replication; therefore, patients with hepatitis D tend to have lower levels of HBV replication. HDV antigen is expressed primarily in hepatocyte nuclei and is occasionally detectable in serum. During acute HDV infection, anti-HDV of the IgM class predominates, and 30–40 days may elapse after symptoms appear before anti-HDV can be detected. In self-limited infection, anti-HDV is low-titer and transient, rarely remaining detectable beyond the clearance of HBsAg and HDV antigen. In chronic HDV infection, anti-HDV circulates in high titer, and both IgM and IgG anti-HDV can be detected. HDV antigen in the liver and HDV RNA in serum and liver can be detected during HDV replication.
Hepatitis C virus, which, before its identification was labeled “non-A, non-B hepatitis,” is a linear, single-strand, positive-sense, 9600-nucleotide RNA virus, the genome of which is similar in organization to that of flaviviruses and pestiviruses; HCV is the only member of the genus Hepacivirus in the family Flaviviridae. The HCV genome contains a single, large open reading frame (gene) that codes for a virus polyprotein of ~3000 amino acids, which is cleaved after translation to yield 10 viral proteins. The 5′ end of the genome consists of an untranslated region (containing an internal ribosomal entry site, IRES) adjacent to the genes for three structural proteins, the nucleocapsid core protein, C, and two structural envelope glycoproteins, E1 and E2. The 5′ untranslated region and core gene are highly conserved among genotypes, but the envelope proteins are coded for by the hypervariable region, which varies from isolate to isolate and may allow the virus to evade host immunologic containment directed at accessible virus-envelope proteins. The 3′ end of the genome also includes an untranslated region and contains the genes for seven nonstructural (NS) proteins, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. p7 is a membrane ion channel protein necessary for efficient assembly and release of HCV. The NS2 cysteine protease cleaves NS3 from NS2, and the NS3-4A serine protease cleaves all the downstream proteins from the polyprotein. Important NS proteins involved in virus replication include the NS3 helicase; NS3-4A serine protease; the multifunctional membrane-associated phosphoprotein NS5A, an essential component of the viral replication membranous web (along with NS4B); and the NS5B RNA-dependent RNA polymerase (Fig. 360-6). Because HCV does not replicate via a DNA intermediate, it does not integrate into the host genome. Because HCV tends to circulate in relatively low titer, 103−107 virions/mL, visualization of the 50- to 80-nm virus particles remains difficult. Still, the replication rate of HCV is very high, 1012 virions per day; its half-life is 2.7 h. The chimpanzee is a helpful but cumbersome animal model. Although a robust, reproducible, small animal model is lacking, HCV replication has been documented in an immunodeficient mouse model containing explants of human liver and in transgenic mouse and rat models. Although in vitro replication is difficult, replicons in hepatocellular carcinoma–derived cell lines support replication of genetically manipulated, truncated, or full-length HCV RNA (but not intact virions); infectious pseudotyped retroviral HCV particles have been shown to yield functioning envelope proteins. In 2005, complete replication of HCV and intact 55-nm virions were described in cell culture systems. HCV entry into the hepatocyte occurs via the nonliver-specific CD81 receptor and the liver-specific tight junction protein claudin-1. A growing list of additional host receptors to which HCV binds on cell entry includes occludin, low-density lipoprotein receptors, glycosaminoglycans, scavenger receptor B1, and epidermal growth factor receptor, among others. Relying on the same assembly and secretion pathway as low-density and very-low-density lipoproteins, HCV is a lipoviroparticle and masquerades as a lipoprotein, which may limit its visibility to the adaptive immune system and which may explain its ability to evade immune containment and clearance. After viral entry and uncoating, translation is initiated by the IRES on the endoplasmic reticulum membrane, and the HCV polyprotein is cleaved during translation and posttranslationally by host cellular proteases as well as HCV NS2-3 and NS3-4A proteases. Host cofactors involved in HCV replication include cyclophilin A, which binds to NS5A and yields conformational changes required for viral replication, and liver-specific host microRNA miR-122.
Organization of the hepatitis C virus genome and its associated, 3000-amino-acid (AA) proteins. The three structural genes at the 5’ end are the core region, C, which codes for the nucleocapsid, and the envelope regions, E1 and E2, which code for envelope glycoproteins. The 5’ untranslated region and the C region are highly conserved among isolates, whereas the envelope domain E2 contains the hypervariable region. At the 3’ end are seven nonstructural (NS) regions—p7, a membrane protein adjacent to the structural proteins that appears to function as an ion channel; NS2, which codes for a cysteine protease; NS3, which codes for a serine protease and an RNA helicase; NS4 and NS4B; NS5A, a multifunctional membrane-associated phosphoprotein, an essential component of the viral replication membranous web; and NS5B, which codes for an RNA-dependent RNA polymerase. After translation of the entire polyprotein, individual proteins are cleaved by both host and viral proteases.
At least six distinct major genotypes (and a minor genotype 7), as well as >50 subtypes within genotypes, of HCV have been identified by nucleotide sequencing. Genotypes differ from one another in sequence homology by ≥30%, and subtypes differ by approximately 20%. Because divergence of HCV isolates within a genotype or subtype and within the same host may vary insufficiently to define a distinct genotype, these intragenotypic differences are referred to as quasispecies and differ in sequence homology by only a few percent. The genotypic and quasispecies diversity of HCV, resulting from its high mutation rate, interferes with effective humoral immunity. Neutralizing antibodies to HCV have been demonstrated, but they tend to be short lived, and HCV infection does not induce lasting immunity against reinfection with different virus isolates or even the same virus isolate. Thus, neither heterologous nor homologous immunity appears to develop commonly after acute HCV infection. Some HCV genotypes are distributed worldwide, whereas others are more geographically confined (see “Epidemiology and Global Features”). In addition, differences exist among genotypes in responsiveness to antiviral therapy but not in pathogenicity or clinical progression (except for genotype 3, in which hepatic steatosis and clinical progression are more likely).
Currently available, third-generation immunoassays, which incorporate proteins from the core, NS3, and NS5 regions, detect anti-HCV antibodies during acute infection. The most sensitive indicator of HCV infection is the presence of HCV RNA, which requires molecular amplification by PCR or transcription-mediated amplification (TMA) (Fig. 360-7). To allow standardization of the quantification of HCV RNA among laboratories and commercial assays, HCV RNA is reported as international units (IUs) per milliliter; quantitative assays with a broad dynamic range are available that allow detection of HCV RNA with a sensitivity as low as 5 IU/mL. HCV RNA can be detected within a few days of exposure to HCV—well before the appearance of anti-HCV—and tends to persist for the duration of HCV infection. Application of sensitive molecular probes for HCV RNA has revealed the presence of replicative HCV in peripheral blood lymphocytes of infected persons; however, as is the case for HBV in lymphocytes, the clinical relevance of HCV lymphocyte infection is not known.
Scheme of typical laboratory features during acute hepatitis C progressing to chronicity. Hepatitis C virus (HCV) RNA is the first detectable event, preceding alanine aminotransferase (ALT) elevation and the appearance of anti-HCV.
Previously labeled epidemic or enterically transmitted non-A, non-B hepatitis, HEV is an enterically transmitted virus that causes clinically apparent hepatitis primarily in India, Asia, Africa, and Central America; in those geographic areas, HEV is the most common cause of acute hepatitis; one-third of the global population appears to have been infected. This agent, with epidemiologic features resembling those of hepatitis A, is a 27- to 34-nm, nonenveloped, HAV-like virus with a 7200-nucleotide, single-strand, positive-sense RNA genome. HEV has three open reading frames (ORF) (genes), the largest of which, ORF1, encodes nonstructural proteins involved in virus replication. A middle-sized gene, ORF2, encodes the nucleocapsid protein, the major nonstructural protein, and the smallest, ORF3, encodes a structural protein whose function remains undetermined. All HEV isolates appear to belong to a single serotype, despite genomic heterogeneity of up to 25% and the existence of five genotypes, only four of which have been detected in humans; genotypes 1 and 2 appear to be more virulent, whereas genotypes 3 and 4 are more attenuated and account for subclinical infections. Contributing to the perpetuation of this virus are animal reservoirs, most notably in swine. No genomic or antigenic homology, however, exists between HEV and HAV or other picornaviruses; and HEV, although resembling caliciviruses, is sufficiently distinct from any known agent to merit its own classification as a unique genus, Hepevirus, within the family Hepeviridae. The virus has been detected in stool, bile, and liver and is excreted in the stool during the late incubation period. Both IgM anti-HEV during early acute infection and IgG anti-HEV predominating after the first 3 months can be detected. Currently, availability and reliability of serologic/virologic testing for HEV infection is limited but can be done in specialized laboratories (e.g., the Centers for Disease Control and Prevention).