Hepatitis Viruses
Arie J. Zuckerman
General Concepts
Viral hepatitis has emerged as a major public health problem throughout the world affecting several hundreds of millions of people. Viral hepatitis is a cause of considerable morbidity and mortality in the human population, both from acute infection and chronic sequelae which include, in the case of hepatitis B, C and D, chronic active hepatitis and cirrhosis. Hepatocellular carcinoma which is one of the ten most common cancers worldwide, is closely associated with hepatitis B, and at least in some regions of the world with hepatitis C virus.
The hepatitis viruses include a range of unrelated and often highly unusual human pathogens.
Hepatitis A virus
Hepatitis A virus (HAV), classified as hepatovirus, is a small, unenveloped symmetrical RNA virus which shares many of the characteristics of the picornavirus family, and is the cause of infectious or epidemic hepatitis transmitted by the fecal-oral route.
Hepatitis B virus
Hepatitis B virus (HBV), a member of the hepadnavirus group, double-stranded DNA viruses which replicate, unusually, by reverse transcription. Hepatitis B virus is endemic in the human population and hyperendemic in many parts of the world. A number of variants of this virus have been described. Natural hepadna virus infections also occur in other mammals including woodchucks, beechy ground squirrels and ducks.
Hepatitis C virus
Hepatitis C virus (HCV), is an enveloped single-stranded RNA virus which appears to be distantly related (possibly in its evolution) to flaviviruses, although hepatitis C is not transmitted by arthropod vectors. Several genotypes have been identified. Infection with this more recently identified virus is common in many countries. Hepatitis C virus is associated with chronic liver disease and also with primary liver cancer in some countries.
Hepatitis D virus
Hepatitis D virus (HDV) is an unusual, single-stranded, circular RNA virus with a number of similarities to certain plant viral satellites and viroids. This virus requires hepadna virus helper functions for propagation in hepatocytes, and is an important cause of acute and severe chronic liver damage in many regions of the world.
Hepatitis E virus
Hepatitis E virus (HEV), the cause of enterically-transmitted non-A, non-B hepatitis, is another non-enveloped, single-stranded RNA virus, which shares many biophysical and biochemical features with caliciviruses. The most similar genome to HEV is found in a plant virus, beet necrotic yellow vein virus, and there are similarities in the functional domains to rubella virus. Final taxonomic classification is yet to be agreed upon.
Hepatitis E virus is an important cause of large epidemics of acute hepatitis in the subcontinent of India, Central and Southeast Asia, the Middle East, parts of Africa and elsewhere. This virus is responsible for high mortality (15-20%), during pregnancy particularly during the third trimester.
The GB hepatitis viruses
The GB hepatitis viruses (GBV-A, GBV-B and GBV-C). The GB hepatitis viruses were cloned recently and preliminary genomic characterization shows that they are related to other positive-stranded RNA viruses with local regions of sequence identity with various flaviviruses. Phylogenetic analysis of genomic sequences showed that these viruses are not genotypes of the hepatitis C virus.
Hepatitis A
Outbreaks of jaundice have been frequently described for many centuries and the term infectious hepatitis was coined in 1912 to describe the epidemic form of the disease. Hepatitis A virus (HAV) is spread by the fecal-oral route and continues to be endemic throughout the world and hyperendemic in areas with poor standards of sanitation and hygiene. The seroprevalence of antibodies to HAV has declined since World War II in many countries, but large epidemics do occur. For example, an outbreak of hepatitis A associated with the consumption of clams in Shanghai in 1988 resulted in almost 300,000 cases.
Hepatitis A has an incubation period of about four weeks. The virus replicates in the liver. Relatively large quantities of virus are shed in the feces during the incubation period before the onset of clinical symptoms, and a brief viremia occurs. The severity of illness ranges from the asymptomatic to anicteric or icteric hepatitis. The virus is non-cytopathic when grown in cell culture. Its pathogenicity in vivo, which involves necrosis of parenchymal cells and histiocytic periportal inflammation, may be mediated by cellular immune responses. By the time of onset of symptoms, excretion of virus in the feces has declined and may have ceased and anti-HAV IgM increases in titer. Anti-HAV IgG may be detected one to two weeks later and persists for years.
Distinctive properties
Electron microscopic examination of concentrates of filtered fecal extracts from patients during the early stages of infection reveals 27 nm icosahedral particles typical of the Picornaviridae (Fig.70-1). HAV was classified in 1983 in the genus Enterovirus (as enterovirus 72) of the family Picornaviridae, on the basis of its biophysical and biochemical characteristics, including stability at low pH. After the entire nucleotide sequence of the viral genome was determined, comparison with other picornavirus sequences revealed limited homology to the enteroviruses, and the virus is now considered as a separate genus, hepatovirus.
FIGURE 70-1 Hepatitis A virus particles found in fecal extracts by immunoelectron microscopy. Both full and empty particles are present. The virus is 27 to 29 nm in diameter. (X 125,000.)
The HAV genome comprises about 7,500 nucleotides (nt) of positive sense RNA which is polyadenylated at the 3' end and has a polypeptide (VPg) attached to the 5' end. A single, large open reading frame (ORF) occupies most of the genome and encodes a polyprotein with a theoretical molecular mass of Mr 252,000. The HAV polyprotein is processed to yield the structural (located at the amino-terminal end) and non-structural viral polypeptides. Many of the features of replication of the picornaviruses have been deduced from studies of prototype enteroviruses and rhinoviruses, in particular poliovirus type 1.
Pathogenesis
The clinical expression of infection with hepatitis A virus varies considerably, ranging from subclinical, anicteric, and mild illnesses in children to the full range of symptoms with jaundice in adults. The ratio of anicteric to icteric illnesses varies widely, both in individual cases and in outbreaks.
Hepatitis A virus enters the body by ingestion and intestinal infection. The virus then spreads, probably by the bloodstream, to the liver, a target organ. Large numbers of virus particles are detectable in feces during the incubation period, beginning as early as 10-14 days after exposure and continuing, in general, until peak elevation of serum aminotransferases. Virus is also detected in feces early in the acute phase of illness, but relatively infrequently after the onset of clinical jaundice. Interestingly, antibody to hepatitis A virus that persists is also detectable late in the incubation period, coinciding approximately with the onset of biochemical evidence of liver damage. Hepatitis A antigen has been localized by immunofluorescence in the cytoplasm of hepatocytes after experimental transmission to chimpanzees. The antigen has not been found in any tissue other than the liver following intravenous inoculation.
Pathological changes produced by hepatitis A appear exclusively in the liver. Several such changes occur: conspicuous focal activation of sinusoidal lining cells; accumulations of lymphocytes and more histiocytes in the parenchyma, often replacing hepatocytes lost by cytolytic necrosis predominantly in the periportal areas; occasional coagulative necrosis in the form of acidophilic bodies; and focal degeneration.
Host Defenses
Antibody to hepatitis A virus develops late in the incubation period. Specific hepatitis A IgM is found in the serum within 1 week from the onset of dark urine, reaching maximum levels after 1 week and declining slowly during the next 40-60 days. Specific IgG antibody appears shortly after IgM is detectable, reaching a maximum titer after 60-80 days. This antibody is protective and persists for many years.
Epidemiology
Viral hepatitis type A (previously named infectious or epidemic hepatitis) occurs endemically in all parts of the world, with frequent reports of minor and major outbreaks. The exact incidence is difficult to estimate because of the high proportion of subclinical infections and infections without jaundice, differences in surveillance, and differing patterns of disease. The degree of under reporting is believed to be very high.
The incubation period of hepatitis A is 3-5 weeks, with a mean of 28 days. Subclinical and anicteric cases are common and, although the disease has in general a low mortality rate, patients may be incapacitated for many weeks. There is no evidence of progression to chronic liver damage.
Hepatitis A virus is spread by the fecal-oral route, person-to-person contact; and under conditions of poor sanitation and overcrowding. Common source outbreaks are most frequently initiated by fecal contamination of water and food, but waterborne transmission is not a major factor in maintaining this infection in industrialized communities. On the other hand, many food-borne outbreaks have been reported. This can be attributed to the shedding of large quantities of virus in the feces during the incubation period of the illness in infected food handlers; the source of the outbreak often can be traced to uncooked food or food that has been handled after cooking. Although hepatitis A remains endemic and common in the developed countries, the infection occurs mainly in small clusters, often with only few identified cases.
In 1973, immune electron microscopy led to the identification of virus particles in extracts of feces (Fig. 70-2) during the early acute phase of illness, providing the long-awaited lead to further studies of this infection. Availability of viral antigen permitted, in turn, identification of specific antibody, development of serologic tests for hepatitis A, and determination of susceptibility to infection in human and nonhuman primates. Human hepatitis A has been transmitted to certain species of non-human primates shown to be free of homologous antibody, thereby providing a model for experimental infection and, initially, also a source of reagents.
FIGURE 70-2 Immune aggregate of hepatitis A virus following the addition of convalescent serum to a fecal extract during the acute phase of the illness X 400,000 (from a series by Anthea Thornton and A J Zuckerman).
The availability of specific serologic tests for hepatitis A made possible the study of the incidence and distribution of hepatitis A in various countries. These studies have shown that infections with hepatitis A virus are widespread and endemic in all parts of the world, chronic excretion of hepatitis A virus does not occur, the infection is rarely transmitted by blood transfusion, and no evidence of progression to chronic liver disease has been found.
Diagnosis
Various serologic tests are available for hepatitis A, including immune electron microscopy, complement-fixation, immune adherence hemagglutination, radioimmunoassay, and enzyme immunoassay. Immune adherence hemagglutination, which had been widely used, is moderately specific and sensitive. Several methods of radioimmunoassay have been described; of these, a solid-phase type of assay is particularly convenient, very sensitive, and specific. Very sensitive enzyme immunoassay techniques are used widely.
Only one serotype of hepatitis A virus has been identified in volunteers infected experimentally with the MS-1 strain of hepatitis A, in patients from different outbreaks of hepatitis in different geographic regions, and in random cases of hepatitis A.
Isolation of virus in tissue culture requires prolonged adaptation and it is, therefore, not suitable for diagnosis.
Control and Prevention of Hepatitis A
In areas of high prevalence, most children are infected early in life and such infections are generally asymptomatic. Infections acquired later in life are of increasing clinical severity. Less than 10% of cases of acute hepatitis A in children up to the age of six are icteric, but this increases to 40-50% in the 6-14 age group and to 70-80% in adults.
Of 115,551 cases of hepatitis A in the USA between 1983 and 1987, only 9% of the cases, but more than 70% of the fatalities, were in those aged over 49. It is important, therefore, to protect those at risk because of personal contact with infected individuals or because of travel to a highly endemic area. Other groups at risk of hepatitis A infection include staff and residents of institutions for the mentally handicapped, day care centers for children, sexually active male homosexuals, intravenous drug abusers, sewage workers, certain groups of health care workers such as medical students on elective studies in countries where hepatitis A is common, military personnel, and certain low socio-economic groups in defined community settings. Patients with chronic liver disease, especially if visiting an endemic area, should be immunized against hepatitis A. In some developing countries, the incidence of clinical hepatitis A is increasing as improvements in socio-economic conditions result in infection later in life, and strategies for immunization are yet to be developed and agreed.
Passive protection may be obtained by the administration of pooled normal human immunoglobulin, containing at least 100 IU/ml of anti-HAV given intramuscularly at a dose of 2 IU/kg body weight. Post-exposure prophylaxis, if given early enough, may prevent or attenuate a clinical illness.
Inactivated hepatitis A vaccines have been developed over the past decade and are now licensed in many countries. The virus grows poorly in cell culture but yields have been improved by adaptation and are sufficient to permit gradient purification. This virus is inactivated with formaldehyde and the antigen adsorbed to aluminum hydroxide and given intramuscularly. The preparations are safe and highly immunogenic in man and have been shown to induce a protective immune response in susceptible non-human primates and during extensive clinical trials in man.
Attenuated strains of HAV have been developed and may be useful potentially as vaccines. This approach is attractive because live vaccines are cheaper to produce and tend to mimic the antibody response induced by natural infection. As with vaccine strains of polioviruses, attenuation may be associated with mutations in the 5' non-coding region of the genome which affect secondary structure. There is also evidence that mutations in the region of the genome encoding the non-structural polypeptides may be important for adaptation to cell culture and attenuation. However, markers of attenuation of HAV have not been identified. Excretion in feces occurs and reversion to virulence may also be a problem. On the other hand, there is also concern that "over-attenuated" viruses may not be sufficiently immunogenic.
Hepatitis E
Retrospective testing of serum samples from patients involved in various epidemics of hepatitis associated with contamination of water supplies with human feces indicated that an agent other than HAV (or hepatitis B) was involved. Epidemics of enterically transmitted non-A, non-B hepatitis in the Indian subcontinent were first reported in 1980, but outbreaks involving tens of thousands of cases have also been documented in the USSR, Southeast Asia, Northern Africa, Mexico and previously in India. The average incubation period is slightly longer than for hepatitis A, with a mean of six weeks. The highest attack rates are found in young adults, and high mortality rates of up to 20% have been reported in women during pregnancy.
Virus-like particles have been detected in the feces of infected individuals by immune electron microscopy using convalescent serum. However, such studies have often proved inconclusive, and a large proportion of the excreted virus may be degraded during passage through the gut. The particles have a mean diameter of 32-34 nm. Cross reaction studies between sera and virus in feces associated with a variety of epidemics in several different countries suggests that a single serotype of virus is involved.
Studies on hepatitis E virus (HEV) have progressed following transmission to susceptible non-human primates. HEV was first transmitted to cynomolgus macaques and a number of other species of monkeys, including chimpanzees, also have been infected. Attempts to amplify the virus by replication in cell culture have been unsuccessful.
Hepatitis E virus was cloned in 1991 and the entire 7.5 kb sequence is known. The organization of the genome is distinct from the Picornaviridae and the non-structural and structural polypeptides are encoded respectively at the 5' and 3' ends. HEV resembles the caliciviruses in the size and organization of its genome, as well as the size and morphology of the virion.
Sequencing of the HEV genome has allowed the development of a number of specific diagnostic tests. For example, HEV RNA was detected, using the polymerase chain reaction (PCR), in fecal samples obtained during a recent epidemic in Kanpur (North India). An enzyme, immunoabsorbent assay, which detects both IgG and IgM anti-HEV, has been developed using a recombinant HEV-glutathione-S-transferase fusion protein and used to detect antibodies in sporadic cases of enterically-transmitted non-A, non-B hepatitis in children in Egypt.
Preliminary but significant progress has been made towards the development of hepatitis E vaccine, using the trpE-C2 fusion protein. In limited experiments, 3 doses of the fusion protein, which represents the carboxyl two-thirds of the putative capsid protein, prevented the development of biochemical evidence of hepatitis after challenge with wild-type virus.
Hepatitis B
Hepatitis B virus was originally recognized as the agent responsible for "serum hepatitis", the most common form of parenterally transmitted viral hepatitis, and an important cause of acute and chronic infection of the liver. The incubation period of hepatitis B is variable with a range of 1 to 6 months. The clinical features of acute infection resemble those of the other viral hepatitides. Acute hepatitis B is frequently anicteric and asymptomatic, although a severe illness with jaundice can occur and occasionally acute liver failure may develop.
Distinctive properties
The virus persists in 5 to 10% of immunocompetent adults, and in as many as 90% of infants infected perinatally. Persistent carriage of hepatitis B, defined by the presence of hepatitis B surface antigen (HBsAg) in the serum for more than six months, have been estimated to affect about 350 million people worldwide. The pathology is mediated by the responses of the cellular immune response of the host to the infected hepatocytes. Long term continuing virus replication may lead to progression to cirrhosis and hepatocellular carcinoma.
In the first phase of chronicity, virus replication continues in the liver, and replicative intermediates of the viral genome may be detected in DNA extracted from liver biopsies. Markers of virus replication in serum include HBV DNA, the S1 proteins (HBsAg) and a soluble antigen, hepatitis B e antigen (HBeAg) which is secreted by infected hepatocytes. In those infected at a very young age, this phase may persist for life but, more usually, virus levels decline over time. Eventually, in most individuals, there is immune clearance of infected hepatocytes associated with seroconversion from HBeAg to anti-HBe.
During the period of replication, the viral genome may integrate into the chromosomal DNA of some hepatocytes and these cells may persist and expand clonally. Rarely does seroconversion to anti-HBs follow clearance of virus replication but, more frequently, HBsAg persists during a second phase of chronicity as a result of the expression of integrated viral DNA.
Structure of the Virus
The hepatitis B virion is a 42-nm particle comprising an electron-dense core (nucleocapsid) 27 nm in diameter surrounded by an outer envelope of the surface protein (HBsAg) embedded in membranous lipid derived from the host cell (Fig. 70-3). The surface antigen is produced in excess by the infected hepatocytes and is secreted in the form of 22-nm particles and tubular structures of the same diameter (initially referred to as Australia antigen).
FIGURE 70-3 Electron micrograph of serum containing hepatitis B virus after negative staining. The three morphologic forms are shown intermingled in this photograph: small pleomorphic spherical particles 20 to 22 nm in diameter; tublar forms; 42nm double-shelled virus. (X400.00)
The 22 nm particles are composed of the major surface protein in both non-glycosylated (p 24) and glycosylated (gp 27) form in approximately equimolar amounts, together with a minority component of the so-called middle proteins (gp 33 and gp 36) which contain the pre-S2 domain, a glycosylated 55 amino acid N-terminal extension. The surface of the virion has a similar composition but also contains the large surface proteins (p 39 and gp 42), which include both the pre-S1 and pre-S2 regions. These large surface proteins are not found in the 22 nm spherical particles (but may be present in the tubular forms in highly viremic individuals) and their detection in serum correlates with viremia. The domain which binds to the specific HBV receptor on the hepatocyte is believed to reside within the pre-S1 region.
The nucleocapsid of the virion consists of the viral genome surrounded by the core antigen (HBcAg). The genome, which is approximately 3.2 kilobases in length, has an unusual structure and is composed of two linear strands of DNA held in a circular configuration by base-pairing at the 5' ends. One of the strands is incomplete and the 3' end is associated with a DNA polymerase molecule which is able to complete that strand when supplied with deoxynucleoside triphosphates.
Organization of the HBV Genome
The genomes of more than a dozen isolates of hepatitis B virus have been cloned and the complete nucleotide sequences determined. Analysis of the coding potential of the genome reveals four open reading frames (ORFs) which are conserved between all of these isolates.
The first ORF encodes the various forms of the surface protein and contains three in-frame methionine codons which are used for initiation of translation. A second promoter is located upstream of the pre-S1 initiation codon. This directs the synthesis of a 2.4 kb mRNA which is co-terminal with the other surface messages and is translated to yield the large (pre-S1) surface proteins.
The core open reading frame also has two in-phase initiation codons. The "precore" region is highly conserved, has the properties of a signal sequence and is responsible for the secretion of HBeAg.
The third ORF, which is the largest and overlaps the other three, encodes the viral polymerase. This protein appears to be another translation product of the 3.5 kb RNA, and is synthesized apparently following internal initiation of the ribosome.
The amino terminal domain is believed to be the protein primer for minus strand synthesis. There is then a spacer region followed by the (RNA and DNA-dependent) DNA polymerase.
The fourth ORF was designated "x" because the function of its small gene product was not known. However, "x" has now been demonstrated to be a transcriptional transactivator (Fig.70-4).
FIGURE 70-4 Structure and genomic organization of hepatitis B virus.
Host Defenses
Antibody and cell-mediated immune responses to various types of antigens are induced during the infection. However, these do not always seem to be protective and, in some instances, may cause autoimmune phenomena that contribute to disease pathogenesis. The immune response to infection with hepatitis B virus is directed toward at least three antigens: hepatitis B surface antigen, the core antigen, and the e antigen. The view that hepatitis B exerts its damaging effect on hepatocytes by direct cytopathic changes is inconsistent with the persistence of large quantities of surface antigen in liver cells of many apparently healthy persons who are carriers. Additional evidence suggests that the pathogenesis of liver damage in the course of hepatitis B infection is related to the immune response by the host.
The surface antigen appears in the sera of most patients during the incubation period, 2-8 weeks before biochemical evidence of liver damage or onset of jaundice. The antigen persists during the acute illness and usually clears from the circulation during convalescence. Next to appear in the circulation is the virus-associated DNA polymerase activity, which correlates in time with damage to liver cells as indicated by elevated serum transaminases. The polymerase activity persists for days or weeks in acute cases and for months or years in some persistent carriers. Antibody to the core antigen is found in the serum 2-10 weeks after the surface antigen appears, and it is frequently detectable for many years after recovery. The titer of core antibody appears to correlate with the amount and duration of virus replication. Finally, antibody to the surface antigen component appears.
During the incubation period and during the acute phase of the illness, surface antigen-antibody complexes may be found in the sera of some patients. Immune complexes have been found by electron microscopy in the sera of all patients with fulminant hepatitis, but are seen only infrequently in nonfulminant infection. Immune complexes also are important in the pathogenesis of other disease syndromes characterized by severe damage of blood vessels (for example, polyarteritis nodosa, some forms of chronic glomerulo-nephritits, and infantile papular acrodermatitis).
Immune complexes have been identified in variable proportions of patients with virtually all the recognized chronic sequelae of acute hepatitis. Deposits of such immune complexes have also been demonstrated in the cytoplasm and plasma membrane of hepatocytes and on or in the nuclei; why only a small proportion of patients with circulating complexes develop vasculitis or polyarteritis is, however, not clear. Perhaps complexes are critical pathogenic factors only if they are of a particular size and of a certain antigen-to-antibody ratio.
Cellular immune responses are known to be particularly important in determining the clinical features and course of viral infections. The occurrence of cell-mediated immunity to hepatitis B antigens has been demonstrated in most patients during the acute phase of hepatitis B and in a significant proportion of patients with surface-antigen-positive chronic active hepatitis, but not in asymptomatic persistent hepatitis B carriers. These observations suggest that cell-mediated immunity may be important in terminating the infection and, under certain circumstances, in promoting immune-mediated liver damage and in the genesis of autoimmunity. Also, evidence suggests that progressive liver damage may result from an autoimmune reaction directed against hepatocyte membrane antigens, initiated in many cases by infection with hepatitis B virus. Although exogenous interferon may be effective in treating some patients with chronic hepatitis, as yet endogenous interferon production has not been detected during the natural infection. More studies to define the role of interferon are needed.
Epidemiology
Although various body fluids (blood, saliva, menstrual and vaginal discharges, serous exudates, seminal fluid, and breast milk) have been implicated in the spread of infection, infectivity appears to be especially related to blood. The epidemiologic propensities of this infection are, therefore, wide. They include infection by inadequately sterilized syringes and instruments, transmission by unscreened blood transfusion and blood products, by close contact, and by sexual contact. Antenatal (rarely) and perinatal (frequently) transmission of hepatitis B infection from mother to child may take place; in some parts of the world (Southeast Asia and Japan) Table 70-1, perinatal transmission is very common.
Diagnosis
Direct demonstration of virus in serum samples is feasible by visualizing the virus particles by electron microscopy, by detecting virus-associated DNA polymerase, and by assay of viral DNA.
All of these direct techniques are impractical under general diagnostic laboratory conditions, and specific diagnoses must therefore rely on serologic tests (Table 70-2).
Hepatitis B surface antigen first appears during the late stages of the incubation period and is easily detectable by radioimmunoassay or enzyme immunoassay. The antigen persists during the acute phase of the disease and sharply decreases when antibody to the surface antigen becomes detectable. Antibody of the IgM class to the core antigen is found in the serum after the onset of the clinical symptoms and slowly declines after recovery. Its persistence at high titer suggests continuation of the infection. Core antibody of the IgG class persists for many years and provides evidence of past infection.
Protection of hepatitis B
The discovery of variation in the epitopes presented on the surface of the virions and subviral particles identified several subtypes of HBV which differ in their geographical distribution. All isolates of the virus share a common epitope, a, which is a domain of the major surface protein which is believed to protrude as a double loop from the surface of the particle. Two other pairs of mutually exclusive antigenic determinants, d or y and w or r, are also present on the major surface protein. These variations have been correlated with single nucleotide changes in the surface ORF which lead to variation in single amino acids in the protein. Four principal subtypes of HBV are recognized: adw, adr, ayw and ayr. Subtype adw predominates in northern Europe, the Americas and Australasia and also is found in Africa and Asia. Subtype ayw is found in the Mediterranean region, eastern Europe, northern and western Africa, the near East and the Indian subcontinent. In the Far East, adr predominates. But the rarer ayr occasionally may be found in Japan and Papua New Guinea.
The major response of recipients of hepatitis B vaccine is to the common a epitope with consequent protection against all subtypes of the virus. First generation vaccines were prepared from 22 nm HBsAg particles purified from plasma donations from chronic carriers. These preparations are safe and immunogenic but have been superseded in some countries by recombinant vaccines produced by the expression of HBsAg in yeast cells. The expression plasmid contains only the 3' portion of the HBV surface ORF and only the major surface protein, without pre-S epitopes, is produced. Vaccines containing pre-S2 and pre-S1, as well as the major surface proteins expressed by recombinant DNA technology, are undergoing clinical trials.
In many areas of the world with a high prevalence of HBsAg carriage, such as China and Southeast Asia, the predominant route of transmission is perinatal. Although HBV does not usually cross the placenta, the infants of viremic mothers have a very high risk of infection at the time of birth. Administration of a course of vaccine with the first dose immediately after birth is effective in preventing transmission from an HBeAg-positive mother in approximately 70% of cases, and this protective efficacy rate may be increased to greater than 90% if the vaccine is accompanied by the simultaneous administration of hepatitis B immune globulin (HBIG).
Immunization against hepatitis B is now recognized as a high priority in preventive medicine in all countries and strategies for immunization are being revised and universal vaccination of infants and adolescents is under examination as a possible strategy to control the transmission of this infection. About 30 countries including the United States now offer hepatitis B vaccine to all children.
However, immunization against hepatitis B is at present recommended in a number of countries with a low prevalence of hepatitis B only to groups which are at an increased risk of acquiring this infection. These groups include individuals requiring repeated transfusions of blood or blood products, prolonged in-patient treatment, patients who require frequent tissue penetration or need repeated access to the circulation, patients with natural or acquired immune deficiency and patients with malignant diseases. Viral hepatitis is an occupational hazard among health care personnel and the staff of institutions for the mentally retarded, and those in some semi-closed institutions. High rates of infection with hepatitis B occur in intravenous drug abusers, sexually active male homosexuals and prostitutes. Individuals working in high endemic areas are, however, at an increased risk of infections and should be immunized. Young infants, children and susceptible persons (including travellers) living in certain tropical and sub-tropical areas where present socio-economic conditions are poor and the prevalence of hepatitis B is high, should also be immunized.
Hepatitis B Antibody Escape Mutants
Production of antibodies to the group antigenic determinant a mediates cross-protection against all sub-types, as has been demonstrated by challenge with a second subtype of the virus following recovery from an initial experimental infection. The epitope a is located in the region of amino acids 124-148 of the major surface protein, and appears to have a double-loop conformation. A monoclonal antibody which recognizes a region within this a epitope is capable of neutralizing the infectivity of hepatitis B virus for chimpanzees, and competitive inhibition assays using the same monoclonal antibody demonstrate that equivalent antibodies are present in the sera of subjects immunized with either plasma-derived or recombinant hepatitis B vaccine.
During a study of the immunogenicity and efficacy of hepatitis B vaccines in Italy, a number of individuals who had apparently mounted a successful immune response and become anti-surface antibody (anti-HBs)-positive, later became infected with HBV.
These cases were characterized by the co-existence of non-complexed anti-HBs and HBsAg, and in 32 of 44 vaccinated subjects there were other markers of hepatitis B infection. Furthermore, analysis of the antigen using monoclonal antibodies suggested that the a epitope was either absent or masked by antibody. Subsequent sequence analysis of the virus from one of these cases revealed a mutation in the nucleotide sequence encoding the a epitope, the consequence of which was a substitution of arginine for glycine at amino acid position 145.
There is now considerable evidence for a wide geographical distribution of the point mutation in hepatitis B virus from guanosine to adenosine at position 587, resulting in an amino acid substitution at position 145 from glycine to arginine in the highly antigenic group determinant a of the surface antigen. This stable mutation has been found in viral isolates from children several years later and it has been described in Italy, Singapore, Japan, and Brunei, and from liver transplant recipients with hepatitis B in the US, Germany, and the UK who had been treated with specific hepatitis B immunoglobulin or humanized hepatitis B monoclonal antibody.
The region in which this mutation occurs is an important virus epitope to which vaccine-induced neutralizing antibody binds, as discussed above, and the mutant virus is not neutralized by antibody to this specificity. It can replicate as a competent virus, implying that the amino acid substitution does not alter the attachment of the virus to the liver cell.
Variants of HBV with altered antigenicity of the envelope protein show that HBV is not as antigenically singular as previously believed and that humoral escape mutation can occur in vivo. There are two causes for concern: failure to detect HBsAg may lead to transmission through donated blood or organs, and HBV may infect individuals who are anti-HBs positive after immunization. Variation in the second loop of the a determinant seems especially important. Mutants, variants, altered genotypes, and unusual strains are now being sought in many laboratories.
HBV Precore mutants
The nucleotide sequence of the genome of a strain of HBV cloned from the serum of a naturally infected chimpanzee has been reported. A surprising feature was a point mutation in the penultimate codon of the precore region which changed the tryptophan codon (TGG) to an amber termination codon (TAG). The nucleotide sequence of the HBV precore region from a number of anti-HBe-positive Greek patients was investigated by direct sequencing PCR-amplified HBV DNA from serum. An identical mutation of the penultimate codon of the precore region to a termination codon was found in seven of eight anti-HBe positive patients who were positive for HBV DNA in serum by hybridization. In most cases there was an additional mutation in the proceeding codon. Similar variants were found by amplification of HBV DNA from serum of anti-HBe positive patients in Italy and Greece. These variants are not confined to the Mediterranean region. The same nonsense mutation (without a second mutation in the adjacent codon) has been observed in patients from Japan and elsewhere, along with rarer examples of defective precore regions caused by frameshifts or loss of the initiation codon for the precore region.
In many cases, precore variants have been described in patients with severe chronic liver disease and who may have failed to respond to therapy with interferon. This observation raises the question of whether they are more pathogenic than the wild-type virus.
HBV and Hepatocellular Carcinoma
When tests for HBsAg became widely available, regions of the world where the chronic carrier state is common were found to be coincident with those where there is a high prevalence of primary liver cancer. Furthermore, in these areas, patients with tumor almost invariably are seropositive for HBsAg. A prospective study in Taiwan revealed that 184 cases of hepatocellular carcinoma occurred in 3,454 carriers of HBsAg at the start of the study, but only 10 such tumors arose in the 19,253 control males who were HBsAg negative.
Southern hybridization of tumor DNA yields evidence of chromosomal integration of viral sequences in at least 80% of HCCs from HBsAg carriers. There is no similarity in the pattern of integration between different tumors, and variation is seen both in the integration site(s) and in the number of copies or partial copies of the viral genome. Sequence analysis of the integrants reveals direct repeats in the viral genome often lie close to the virus/cell junctions, suggesting that sequences around the ends of the viral genome may be involved in recombination with host DNA. Integration seems to involve microdeletion of host sequences and rearrangements and deletions of part of the viral genome also may occur. When an intact surface gene is present, the tumor cells may produce and secrete HBsAg in the form of 22 nm particles. Production of HBcAg by tumors is rare, however, and the core ORF is often incomplete and modifications such as methylation may also modulate its expression. Cytotoxic T cells targeted against core gene products on the hepatocyte surface seem to be the major mechanism of clearance of infected cells from the liver, and cells with integrated viral DNA which are capable of expressing these proteins also may be lysed. Thus, there may be immune selection of cells with integrated viral DNA which are incapable of expressing HBcAg.
The mechanisms of oncogenesis by HBV remain obscure. HBV may act non-specifically by stimulating active regeneration and cirrhosis which may be associated with long-term chronicity. However, HBV-associated tumors occasionally arise in the absence of cirrhosis, and such hypotheses do not explain the frequent finding of integrated viral DNA in tumors. In rare instances, the viral genome has been found to be integrated into cellular genes such as cyclin A and a retinoic acid receptor. Translocations and other chromosomal rearrangements also have been observed. Although insertional mutagenesis of HBV remains an attractive hypothesis to explain its oncogenicity, there is insufficient supportive evidence.
Like many other cancers, development of hepatocellular carcinoma is likely to be a multifactorial process. The clonal expansion of cells with integrated viral DNA seems to be an early stage in this process and such clones may accumulate in the liver throughout the period of active virus replication. In areas where the prevalence of primary liver cancer is high, virus infection usually occurs at an early age and virus replication may be prolonged, although the peak incidence of tumor is many years after the initial infection.
Hepatitis D
Delta hepatitis was first recognized following detection of a novel protein, delta antigen (HDAg), by immunofluorescent staining in the nuclei of hepatocytes from patients with chronic active hepatitis B.
Hepatitis delta virus (HDV) is now known to require a helper function of HBV for its transmission. HDV is coated with HBsAg which is needed for release from the host hepatocyte and for entry in the next round of infection.
Two forms of delta hepatitis infection are known. In the first, a susceptible individual is co-infected with HBV and HDV, often leading to a more severe form of acute hepatitis caused by HBV. Vaccination against HBV also prevents co-infection. In the second, an individual chronically infected with HBV becomes superinfected with HDV. This may cause a second episode of clinical hepatitis and accelerate the course of the chronic liver disease, or cause overt disease in asymptomatic HBsAg carriers. HDV itself seems to be cytopathic and HDAg may be directly cytotoxic.
Delta hepatitis is common in some areas of the world with a high prevalence of HBV infection, particularly the Mediterranean region, parts of Eastern Europe, the Middle East, Africa and South America. It has been estimated that 5% of HBsAg carriers worldwide (approximately 15 million people) are infected with HDV. In areas of low prevalence of HBV, those at risk of hepatitis B, particularly intravenous drug abusers, are also at risk of HDV infection.
Distinctive Properties of HDV
The HDV particle is approximately 36 nm in diameter and composed of an RNA genome associated with HDAg, surrounded by an envelope of HBsAg. The HDV genome is a closed circular RNA molecule of 1679 nucleotides and resembles those of the satellite viroids and virusoids of plants and similarly seems to be replicated by the host RNA polymerase II with autocatalytic cleavage and circularization of the progeny genomes via trans-esterification reactions (ribosome activity). Consensus sequences of viroids which are believed to be involved in these processes also are conserved in HDV. Unlike the plant viroids, HDV codes for a protein, HDAg.
This is encoded in an open reading frame in the antigenomic RNA but four other open reading frames which are also present in the genome do not appear to be utilized. The antigen, which contains a nuclear localization signal, was originally detected in the nuclei of infected hepatocytes and may be detected in serum only after stripping off the outer envelope of the virus with detergent.
Hepatitis C
Transmission studies in chimpanzees established that the main agent of parenterally acquired non-A, non-B hepatitis was likely to be an enveloped virus some 30 to 60 nm in diameter. These studies made available a pool of plasma which contained a relatively high titer of the agent. In order to clone the genome, the virus was pelleted from the plasma. Because it was not known whether the genome was DNA or RNA, a denaturation step was included prior to the synthesis of complementary DNA so that either DNA or RNA could serve as a template. The resultant cDNA was then inserted into the bacteriophage expression vector lambda gt 11 and the libraries screened using serum from a patient with chronic non-A, non-B hepatitis. This approach led to the detection of a clone (designated 5-1-1) which was found to bind to antibodies present in the sera of several individuals infected with non-A, non-B hepatitis. This clone was used as a probe to detect a larger, overlapping clone in the same library. It was possible to demonstrate that these sequences hybridized to a positive-sense RNA molecule of around 10,000 nt which was present in the livers of infected chimpanzees but not in uninfected controls. No homologous sequences could be detected in the chimpanzee or human genomes. By employing a "walking" technique, it was possible to use newly detected overlapping clones as hybridization probes, in turn, to detect further virus-specific clones in the library. Thus, clones covering the entire viral genome were assembled and the complete nucleotide sequence determined.
Diagnosis of HCV Infection
Successful cloning of portions of the viral genome permitted the development of new diagnostic tests for infection by the virus. Since the original antigen was detected by antibodies in the serum of an infected patient it was an obvious candidate for the basis of an ELISA to detect anti-HCV antibodies. A larger clone, C100, was assembled from a number of overlapping clones and expressed in yeast as a fusion protein using human superoxide dismutase sequences to facilitate expression, and this fusion protein formed the basis of first generation tests for HCV infection. The 5-1-1 antigen comprises amino acid sequences from the non-structural, NS4, region of the genome and C100 contains both NS3 and NS4 sequences.
It is now known that antibodies to C100 are detected relatively late following an acute infection. Furthermore, the first generation ELISAs were associated with a high rate of false positive reactions when applied to low incidence populations, and there were further problems with some retrospective studies on stored sera. Data based on this test alone should, therefore, be interpreted with caution.
Second generation tests include antigens from the nucleocapsid and further non-structural regions of the genome. The former (C22) is particularly useful and antibodies to the HCV core protein seem to appear relatively early in infection. These second generation tests confirm that HCV is the major cause of parenterally transmitted non-A, non-B hepatitis. Routine testing of blood donations is now in place in many countries and prevalence rates vary from 0.2-0.5% in northern Europe to 1.2-1.5% in southern Europe and Japan. Most of those with antibody have a history of parenteral risk such as a history of transfusion or administration of blood products or of intravenous drug abuse. There is little evidence for sexual or perinatal transmission of HCV and it is not clear what are the natural routes of transmission.
The availability of the nucleotide sequence of HCV made possible the use of the polymerase chain reaction (PCR) as a direct test for the genome of the virus. There is considerable variation in nucleotide sequences among different isolates of HCV, and the 5' non-coding region, which seems to be highly conserved, is the preferred target for the PCR.
Current data suggest that about 80% of infections with HCV progress to chronicity. Histological examination of liver biopsies from asymptomatic HCV-carriers (blood donors) reveals that none has normal histology and that up to 70% have chronic active hepatitis and/or cirrhosis. Whether the virus is cytopathic or whether there is an immunopathological element remains unclear. HCV infection is also associated with progression to primary liver cancer. For example, in Japan, where the incidence of hepatocellular carcinoma has been increasing despite a decrease in the prevalence of HBsAg, HCV is now considered the major risk factor.
There is no DNA intermediate in the replication of the HCV genome or integration of viral nucleic acid and viral pathology may contribute to oncogenesis through cirrhosis and regeneration of liver cells. HCV rarely seems to cause fulminant hepatitis.
Distinctive Properties of HCV
The genome of HCV resembles those of the pestiviruses and flaviviruses in that it comprises around 10,000 nt of positive sense RNA, lacks a 3' polyA tract and has a similar gene organization. It has been proposed that HCV should be the prototype of a third genus in the family Flaviviridae.
All of these genomes contain a single large open reading frame which is translated to yield a polyprotein (of around 3000 amino acids in the case of HCV) from which the viral proteins are derived by post-translational cleavage and other modifications. The amino acid sequence of the nucleocapsid protein seems to be highly conserved among different isolates of HCV. The next domain in the polyprotein also has a signal sequence at its carboxyl-terminus and may be processed in a similar fashion. The product is a glycoprotein which is probably found in the viral envelope and is variably termed E1/S or gp35. The third domain may be cleaved by a protease within the viral polyprotein to yield what is probably a second surface glycoprotein, E2/NS1 or gp70. These glycoproteins have not been visualized in vivo and the molecular sizes are estimated from sequence data and expression studies in vitro. Other post-translational modifications, including further proteolytic cleavages, are possible. These proteins are the focus of considerable interest because of their potential use in tests for the direct detection of viral proteins and for HCV vaccines. Nucleotide sequencing studies reveal that both domains contain hypervariable regions. It is possible that this divergence has been driven by antibody pressure and that these regions specify important immunogenic epitopes.
The non-structural region of the HCV genome is divided into regions NS2 to NS5. In the flaviviruses, NS3 has two functional domains, a protease which is involved in cleavage of the non-structural region of the polyprotein and a helicase which is presumably involved in RNA replication. Motifs within this region of the HCV genome have homology to the appropriate consensus sequences, suggesting similar functions. NS5 seems to be the replicase and contains the gly-asp-asp motif common to viral RNA-dependent RNA polymerases (Fig. 70-5).
FIGURE 70-5 Hepatitis C Viral Genome.
Hepatitis C virus consists of a family of highly related but nevertheless distinct genotypes, numbering at present 6 genotypes and various subtypes with differing geographical distribution, and with a complex nomenclature. The C, NS3 and NS4 domains are the most highly conserved regions of the genome, and therefore these proteins are the most suitable for use as capture antigens for broadly reactive tests for antibodies to HCV. The sequence differences observed between HCV groups suggest that virus-host interactions may be different, which could result in differences in pathogenicity and in response to antiviral therapy. It is important, therefore, to develop group- and virus-specific tests. The degree of divergence apparent within the viral envelope proteins implies the absence of a broad cross-neutralizing antibody response to infection by viruses of different groups.
In addition to the sequence diversity observed between HCV groups, there is considerable sequence heterogeneity among almost all HCV isolates in the N-terminal region of E2/NS1, implying that this region may be under strong immune selection. Indeed, sequence changes within this region may occur during the evolution of disease in individual patients and may play an important role in progression to chronicity.
Vaccine Development
Problems in vaccine development include the sequence diversity between viral groups and the substantial sequence heterogeneity among isolates in the N-terminal region of E2/NS1. Neutralizing antibodies have not been identified so far. The virus has not been cultivated in vitro (cf. Yellow fever flavivirus, which has been cultured and from which vaccines have been prepared). Nevertheless, approaches to vaccine development could be based on techniques used for the development of vaccines against the Flaviviruses and Pestiviruses.
The GB Hepatitis Viruses
About 30 years ago, a series of transmission studies of human viral hepatitis were initiated in small South American tamarins or marmosets, which were chosen because of their very limited contact with man, implying that they were unlikely to have been infected with human viruses. A serum which was obtained on the third day of jaundice from a young surgeon (GB) with jaundice-induced hepatitis in each of four inoculated marmosets and was passaged serially in these animals. These important observations remained controversial until the application recently of modern molecular virological techniques. Preliminary results indicate the identification of two independent viruses, GBV-A and GBV-B, in the infectious plasma of tamarins inoculated with GB.
GBV-A does not replicate in the liver of tamarins, whereas GBV-B causes hepatitis. Cross-challenge experiments showed that infection with the original infectious tamarin inoculum conferred protection from reinfection with GBV-B but not GBV-A. A third virus, GBV-C, was isolated subsequently from a human specimen which was immunoreactive with a GBV-B protein. GBV-C RNA was found in several patients with clinical hepatitis, and shown to have substantial sequence identity to GBV-A.
A series of studies including phylogenetic analysis of genomic sequences showed that GBV-A, B, and C are not genotypes of hepatitis C virus, and that GBV-A and GBV-C are closely related. GBV-A/C and GBV-B and the hepatitis C viruses are members of distinct viral groups. The organization of the genes of the GBV-A, B, and C genomes shows that they are related to other positive-strand RNA viruses with local regions of sequence identity with various flaviviruses. The three GB viruses and HCV share only limited overall amino acid sequence identity.
Diagnostic reagents were prepared with recombinant antigens, and limited testing was carried out in groups of patients, blood donors and other selected individuals: patients with non-A, B, C, D, E hepatitis, multitransfused patients, intravenous drug addicts and other populations with a high incidence of viral hepatitis. Preliminary studies indicated the presence of antibody to each of the GB viruses in 3% to as many as 14%. The development and availability of specific diagnostic reagents will establish the epidemiology of these newly identified viruses, their pathogenic significance in man and their clinical and public health importance.
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