Chapter 13: Hepatitis Viruses

The causes of hepatitis (inflammation of the liver) are varied and include viruses, bacteria, and protozoa, as well as drugs and toxins (eg, isoniazid, carbon tetrachloride, and ethanol). The clinical symptoms and course of acute viral hepatitis can be similar, regardless of etiology, and determination of a specific cause depends primarily on the use of laboratory tests. Hepatitis may be caused by at least five viruses belonging to different virus families, whose major characteristics are summarized in Table 13–1. Non-A, non-B hepatitis is a term previously used to identify cases of hepatitis not due to hepatitis A virus (HAV) or hepatitis B virus (HBV). With the discovery of hepatitis C and E viruses (HCV and HEV, respectively), virtually all the viral etiologies of non-A, non-B hepatitis can be specifically identified. One additional hepatitis virus, hepatitis G virus (HGV), has been identified that is not associated with any clinical disease so far but found in some blood donors as well as some patients who are either infected with HCV or human immunodeficiency virus (HIV). Other viruses, such as Epstein-Barr virus and cytomegalovirus, can cause inflammation of the liver, but hepatitis is not the primary disease caused by them. Yellow fever is also associated with hepatitis, but is described in Chapter 16.

VIROLOGY

Hepatitis A virus (HAV) belongs to the Picornaviridae (picornaviruses) family and Hepatovirus genus. It is an unenveloped (naked capsid), single-stranded, positive-sense RNA virus with a cubic (icosahedral) symmetry and a diameter of 27 nm (Figure 13–1). The genome of HAV is a 7.4 kb positive-sense, single-stranded RNA bound to a protein called VPg, and each capsid unit comprises four proteins, VP1, -2, -3, and -4, which cover the genome and form a naked capsid icosahedral virion. VP1 is the spike of HAV that binds to the receptor on the host cells. There is only one serotype of HAV. This virus possesses several characteristics of enteroviruses; for example, it resists inactivation and is stable at −20°C with low pH. The virus has been successfully cultivated in primary marmoset liver cell cultures and in fetal rhesus monkey kidney cell cultures.

HAV replicates in the cytoplasm, like other positive-sense RNA viruses (Figure 12–1). HAV interacts with the receptor (α₂-macroglobulin) on the target cells (liver cells and few other cell types) and enters via receptor-mediated endocytosis (viropexis). The positive-sense RNA is translated into a polyprotein, which is cleaved into various mature proteins, including RNA-dependent RNA polymerase. RNA-dependent RNA polymerase directs transcription of mRNAs to produce viral proteins as well as replication to make full-length viral genomes. The assembly of the progeny viruses takes place in the cytoplasm after the packaging of viral genomes into HAV capsid proteins. Virions are released upon cell lysis.

HEPATITIS A DISEASE

EPIDEMIOLOGY

Humans appear to be the major natural hosts of HAV. Several other primates (including chimpanzees and marmosets) are susceptible to experimental infection, and natural infections of these animals may occur. The major mode of spread of HAV is person to person by fecal–oral exposure. Transmission through blood transfusion, though possible, is not an important means of spread, but persons with hemophilia who are given plasma products are at risk. High risk of infection is also observed in men who have sex with men, in illicit drug users, and in travelers from the developed countries visiting developing areas of the world. Although most cases of hepatitis A are not linked to a single contaminated source and occur sporadically, outbreaks have been described. The disease is common under conditions of crowding, and it occurs very frequently in nursing home settings and day care centers. A chronic carrier state has not been observed with hepatitis A; perpetuation of the virus in nature presumably depends on sporadic subclinical infections and person-to-person transmission. Outbreaks of hepatitis A have been linked to the ingestion of undercooked seafood, usually shellfish from waters contaminated with human feces. Common-source outbreaks related to other foods, including vegetables as well as contaminated drinking water, have also been reported.

Less than 50% of the general population of the United States now has serologic evidence of HAV infection, and rates have been decreasing since 1970, apparently because of better sanitation, less crowding, and the use of hepatitis A vaccine. In contrast, more than 90% of the adult population in many developing countries shows evidence of previous hepatitis A infection. The risk of clinically evident disease is much higher in infected adults than in children. Patients are most contagious in the 1 to 2 weeks before onset of clinical disease.

PATHOGENESIS

HAV is believed to replicate initially in the enteric mucosa. It can be demonstrated in feces by electron microscopy for 10 to 14 days before onset of disease. In most patients with symptoms of the disease, virus is no longer found in fecal specimens. Multiplication in the intestines is followed by a period of viremia with spread to the liver. The response to replication in the liver consists of lymphoid cell infiltration, necrosis of liver parenchymal cells, and proliferation of Kupffer cells (Figure 13–2). A variable degree of biliary stasis may be present. It is also believed that cytotoxic T lymphocytes (CTLs) damage the hepatocytes. Except in the rare instance of acute hepatic necrosis, the infection is cleared, liver damage is reversed, and HAV does not establish a chronic infection. Initial immune response is the development of HAV-specific IgM antibody followed by appearance of IgG after a few weeks. Detectable levels of IgG antibody to HAV persist indefinitely in serum, and patients with anti-HAV antibodies are immune to reinfection. Although virus-specific IgA has been demonstrated in stool, secretory immunity has not been shown to be important for hepatitis A. The immunopathogenic events associated with HAV infection are shown in Figure 13–3.

CLINICAL ASPECTS

MANIFESTATIONS

In HAV infection, an incubation period of 15 to 45 days (mean 25 days) is usually followed by fever; anorexia (poor appetite); nausea; pain in the right upper abdominal quadrant; and, within several days, jaundice. Dark urine and clay-colored stools may be noticed by the patient 1 to 5 days before the onset of clinical jaundice. The liver is enlarged and tender, and serum aminotransferase and bilirubin levels are elevated as a result of hepatic inflammation and damage. Recovery occurs in days to weeks.

Many persons who have serologic evidence of acute HAV infection are asymptomatic or only mildly ill, without jaundice (anicteric hepatitis A). The infection-to-disease ratio is dependent on age; it may be as high as 20:1 in children and approximately 1:1 in older adults. Almost all cases (99%) of HAV are self-limiting. Chronic hepatitis such as that seen with hepatitis B is very rare. In rare cases, fulminant fatal hepatitis associated with extensive liver necrosis may occur (∼0.1%).

DIAGNOSIS

Antibody to HAV can be detected during early illness, and most patients with symptoms or signs of acute HAV already have detectable antibody in serum. Early antibody responses are predominantly IgM, which can be detected for several weeks and up to several months (Figure 13–3). During convalescence, antibody of the IgG class predominates. The best method for documentation of acute HAV infection is the demonstration of high titers of virus-specific IgM antibody in serum drawn during the acute phase of illness. Because IgG antibody persists indefinitely, its demonstration in a single serum sample is not indicative of recent infection; a rise in titer between acute and convalescent sera must be documented. Immunoelectron microscopic identification of the virus in fecal specimens and isolation of the virus in cell cultures remain research tools.

TREATMENT AND PREVENTION

There is no specific treatment for patients with acute hepatitis A. Supportive measures include adequate nutrition and rest. Avoidance of exposure to contaminated food or water or infected persons are important measures to reduce the risk of hepatitis A infection.

Passive Immunization

Passive (ie, antibody) prophylaxis for hepatitis A has been available for many years. Immune serum globulin (ISG), manufactured from pools of plasma from large segments of the general population, is protective if given before or during the incubation period of the disease. It has been shown to be about 80% to 90% effective in preventing clinically apparent type A hepatitis. In some cases, infection occurs but disease is ameliorated; that is, patients develop anicteric, usually asymptomatic, hepatitis A. At present, ISG should be administered to household and intimate contacts of hepatitis A patients and those known to have eaten uncooked foods prepared or handled by an infected person. When clinical symptoms have appeared, the patient is already producing antibody, and administration of ISG is not indicated.

Active Immunization

Formalin-killed HAV, which is grown in human cell culture, is used as a vaccine that induces antibody titers similar to those of wild-type virus infection, is almost 100% protective, and is now recommended for all children at age 1 and for adults with a high risk of infection. Two doses are given 6 to 12 months apart to achieve long-term protection. In the United States two inactivated HAV vaccines, HAVRIX (GlaxoSmithKline) and VAQTA (Merck & Co) are currently licensed. In addition, a combination vaccine, TWINRIX (GlaxoSmithKline) that contains both HAV and HBV antigens given in three or four doses to adults of age 18 years and above, is also available. Based on scientific evidence that active immunization is as effective as ISG if given shortly after exposure, the guidelines were revised in 2007 in the United States to give hepatitis A vaccine after exposure to prevent infection in healthy individuals of 1 to 40 years of age. More importantly, the rates of HAV infection have declined by 92% in the United States since the vaccine was made available in 1995.

VIROLOGY

STRUCTURE

Hepatitis B virus (HBV) is an enveloped DNA virus belonging to the family Hepadnaviridae (hepadnaviruses). It is unrelated to any other human virus; however, related hepatotropic agents have been identified in woodchucks, ground squirrels, and kangaroos. A schematic of the HBV is illustrated in Figure 13–4. The complete virion is a 42 nm spherical particle that consists of an envelope around a 27 nm core. The core comprises a nucleocapsid that contains the DNA genome.

The viral genome consists of partially double-stranded DNA with a short, single-stranded piece. It comprises 3200 nucleotides, making it the smallest known DNA virus with respect to genome size but capable of encoding surface (envelope) protein (hepatitis B surface antigen [HBsAg]), core (nucleocapsid) protein (hepatitis B core antigen [HBcAg]), DNA polymerase (reverse transcriptase), and HBx protein (a transcriptional activator). Closely associated with the viral DNA is a viral DNA polymerase, which has RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities (reverse transcriptase). Another component of the core is hepatitis B e antigen (HBeAg), which is a low–molecular-weight glycoprotein secreted from the infected cells. The virion has a lipid bilayer envelope containing the HBsAg, which is composed of one major and two other proteins. The complete virus particle is called a Dane particle.

Aggregates of HBsAg are often found in great abundance in serum during infection. They may assume spherical or filamentous shapes with a mean diameter of 22 nm (Figure 13–4). HBV DNA can also be detected in serum and is an indication that infectious virions are present. In infected liver tissue, evidence of HBcAg, HBeAg, and hepatitis B DNA is found in the nuclei of infected hepatocytes, whereas HBsAg is found in cytoplasm.

There are four major serotypes of HBV (adr, adw, ayr, ayw) based on HBsAg antigenic epitopes. Furthermore, there are eight hepatitis B genotypes (A-H) based on nucleotide sequence variation of HBV genome, which may be associated with different clinical outcomes. These genotypes vary in geographic distribution with genotype A primarily found in North America, Northern Europe, India, and Africa; genotypes B and C in Asia; genotype D in Southern Europe, Middle East, and India; genotype E in West and South Africa; genotype F in South and Central America; genotype G in the United States and Europe, and genotype H in Central America and California.

REPLICATION CYCLE

The replication of HBV involves a reverse transcription step, and, as such, is unique among DNA viruses (Figure 13–5). HBV has a specific tropism for the liver. However, the receptor for HBV and the mechanism of viral entry are not known. The attachment or adsorption of HBV to hepatocytes (liver cells) is mediated by the envelope protein (HBsAg) of the virus, probably by binding of HBsAg with polymerized human serum albumin or other serum proteins. After viral entry, the partially double-stranded DNA (incomplete) is transported to the nucleus. The double-stranded DNA is organized as two strands. One, a short strand, is associated with the viral DNA polymerase and is of positive polarity.

The complete or long strand is complementary and thus is of negative polarity. The partially incomplete strand is formed into a complete double-stranded, circular DNA, which is essential before the transcription can take place. Host RNA polymerase directs the transcription of viral mRNAs to encode early proteins, including HBcAg, HBeAg, and viral DNA polymerase as well as full-length RNA (pregenomic RNA). HBsAg is encoded later and associates with the membranes of endoplasmic reticulum or Golgi apparatus. HBcAg forms the core by enclosing the full-length, positive-sense viral pregenomic RNA along with viral DNA polymerase into maturing core particles late in the replication cycle. These full-length RNA strands form a template for a reverse transcription step in which negative-stranded DNA is synthesized. The RNA template strands are then degraded by ribonuclease H activity. A positive-stranded DNA is then synthesized, although this is not completed before virus maturation in which HBsAg-containing membranes of the endoplasmic reticulum or Golgi apparatus are wrapped over the nucleocapsid core, resulting in the variable-length, short, positive DNA strands found in the virions. The virions are released by exocytosis.

HBV DNA has also been found to integrate into the host chromosomes, especially in HBV-infected patients with hepatocellular carcinoma (HCC). However, the significance of integrated HBV DNA in viral replication is not known. Despite extensive attempts, HBV has not been successfully propagated in the laboratory. Humans appear to be the major host; however, as with hepatitis A, infection of subhuman primates has been accomplished experimentally.

HEPATITIS B DISEASE

EPIDEMIOLOGY

Hepatitis B infection is found worldwide, with prevalence rates varying markedly among countries, but a total of approximately 400 million persons (Figure 13–6). Chronic carriers constitute the main reservoir of infection: in some countries, particularly in the Far East, up to 5% to 15% of all persons carry the virus, and most are asymptomatic. About 10% of patients with HIV infection are chronic carriers of HBV.

In the United States, an estimated 1.25 million people are infected with hepatitis B, and 300 000 new cases occur annually. About 300 of these patients die of acute fulminant hepatitis, and 5% to 10% of infected patients become chronic HBV carriers. As many as 4000 people die yearly of hepatitis B-related cirrhosis, and 1000 die of HCC. The virus is spread vertically, parenterally, and by sexual contact. Approximately 50% of infections in the United States are sexually transmitted, and the prevalence of HBsAg in serum is higher in certain populations, such as among men who have sex with men, patients on hemodialysis or immunosuppressive therapy, patients with Down syndrome, and injection drug users. Routine screening of blood donors for HBsAg and antibody to HBcAg (anti-HBcAg) has markedly decreased the incidence of postblood transfusion and postplasma products hepatitis B transmission. Multiple-pool blood products still cause occasional cases. Exposure to hepatitis viruses from direct contact with blood or other body fluids, probably through needlestick injuries, has resulted in a risk of hepatitis B infection in medical personnel. Attack rates are also high in the sexual partners of infected patients.

Hepatitis B infection of infants does not appear to be transplacentally transmitted to the fetus in utero, but is acquired during the birth process by the swallowing of infected blood or fluids or through abrasions. The rate of virus acquisition is high (up to 90%) in infants born to mothers who have acute hepatitis B infection or are carrying HBsAg and HBeAg. Most infants do not develop clinical disease; however, infection in the neonatal period is associated with failure to produce antibody to HBsAg and cell-mediated immune responses probably as a result of an immature immune system, which allows chronic carriage to occur in nearly 90% to 100% of the infected neonates/infants.

HCC has been strongly associated with persistent carriage of HBV by serologic tests and by detection of viral nucleic acid sequences integrated in tumor cell genomes. In many parts of Africa and Asia, primary liver cancer accounts for 20% to 30% of all types of malignancies, but in North and South America and in Europe, it is only 1% to 2%. The estimated risk of developing the malignancy for persons with chronic HBV is increased to between 10-fold and more than 300-fold in different populations. The risk of HCC further increases in patients with chronic hepatitis B infection and high viral loads.

PATHOGENESIS

In the past, hepatitis B was known as posttransfusion hepatitis or as hepatitis associated with the use of illicit parenteral drugs (serum hepatitis). However, over the last few years it has become clear that the major mode of acquisition is through close personal contact with body fluids of infected individuals. HBsAg has been found in most body fluids, including saliva, semen, and cervical secretions. Under experimental conditions, as little as 0.0001 mL of infectious blood has produced infection. Transmission is therefore possible by vehicles such as inadequately sterilized hypodermic needles and instruments used in tattooing and ear piercing.

The factors determining the clinical manifestations of acute hepatitis B are largely unknown; however, some appear to involve immunologic responses of the host. The serum sickness-like rash and arthritis that may precede the development of symptoms and jaundice appear to be related to circulating immune complexes that activate the complement system. In addition, accumulation of these immune complexes in the kidney results in renal damage. Antibody to HBsAg is protective and associated with resolution of the disease. Cellular immunity also may be important in the host response because patients with insufficient T-lymphocyte function have a high incidence of chronic infection with HBV. However, CTLs cause damage to liver by destroying infected cells. Antibody to HBcAg, which appears during infection, is present in chronic carriers with persistent hepatitis B virion production and does not appear to be protective.

The morphologic lesions of acute hepatitis B resemble those of other hepatitis viruses. In chronic active hepatitis B, the continued presence of inflammatory foci of infection results in necrosis of hepatocytes, collapse of the reticular framework of the liver, and progressive fibrosis. The increasing fibrosis can result in the syndrome of postnecrotic hepatic cirrhosis (Figure 13–7).

Integrated hepatitis B viral DNA can be found in nearly all HCCs. The virus has not been shown to possess a transforming gene but may well activate a cellular oncogene. It is also possible that the virus does not play such a direct molecular role in oncogenicity, because the natural history of chronic hepatitis B infection involves cycles of damage or death of liver cells interspersed with periods of intense regenerative hyperplasia. This significantly increases the opportunity for spontaneous mutational changes that may activate cellular oncogenes. HBV transcriptional transactivator protein, HBx, is known to activate the Src kinase, which may influence HBV-induced carcinogenesis. In addition, HBx protein has been shown to interact with tumor suppressor gene, p53, which may play an important role in development of HCC. Whatever the mechanism, the association between chronic viral infection and HCC is clear, and liver cancer is a major cause of disease and death in countries in which chronic hepatitis B infection is common. The proven success of combined active and passive immunization in aborting hepatitis B infection in infancy and childhood makes HCC a potentially preventable disease.

CLINICAL ASPECTS

MANIFESTATIONS

The clinical picture of hepatitis B is highly variable. The incubation period may be as brief as 30 days or as long as 180 days (mean approximately 60-90 days). Acute hepatitis B is usually manifested by the gradual onset of fatigue, loss of appetite, nausea and pain, and fullness in the right upper abdominal quadrant. Early in the course of disease, pain and swelling of the joints and occasional frank arthritis may occur. Some patients develop a rash. With increasing involvement of the liver, there is increasing cholestasis, and hence clay-colored stools, darkening of the urine, and jaundice. Symptoms may persist for several months before finally resolving.

In general, the symptoms associated with acute hepatitis B are more severe and more prolonged than those of hepatitis A; however, anicteric disease and asymptomatic infection occur. The infection-to-disease ratio, which varies according to patient age and method of acquisition, has been estimated to be approximately 3:1. Fulminant hepatitis, leading to extensive liver necrosis and death, develops in less than 1% of the cases. One important difference between hepatitis A and hepatitis B is the development of chronic hepatitis, which occurs in approximately 10% of all patients with hepatitis B infection, with a much higher risk for newborns (~90%), children (~50%), and the immunocompromised. In immunocompetent adults, the strong cellular immune response results in acute hepatitis and only rarely (~1%) in chronic hepatitis. Chronic infection is associated with ongoing replication of virus in the liver and usually with the presence of HBsAg in serum. Chronic hepatitis may lead to cirrhosis, liver failure, or HCC in up to 25% of the patients.

DIAGNOSIS

The nomenclature of hepatitis B antigens and antibodies is shown in Table 13–2 and the sequence of their appearance is shown in Figure 13–8. During the acute episode of disease, when there is active viral replication, large amounts of HBsAg and HBV DNA can be detected in the serum, as can fully developed virions and high levels of DNA polymerase and HBeAg. Although HBcAg is also present, antibody against it invariably occurs and prevents its detection. Upon resolution of acute hepatitis B, HBsAg and HBeAg disappear from serum with the development of antibodies (anti-HBs and anti-HBe) against them. The development of anti-HBs is associated with elimination of infection and protection against reinfection. Anti-HBc is detected early in the course of disease and persists in serum for years. It is an excellent epidemiologic marker of infection, but is not protective. The laboratory diagnosis of acute hepatitis B is best made by demonstrating the IgM antibody to HBcAg in serum, since this antibody disappears within 6 to 12 months of the acute infection. Almost all patients who develop jaundice are anti-HBc IgM-positive at the time of clinical presentation. HBsAg may also be detected in serum. Past infection with hepatitis B is best determined by detecting IgG anti-HBc, anti-HBs, or both, whereas vaccine induces only anti-HBs.

In patients with chronic hepatitis B, evidence of viral persistence can be found in serum (Figure 13–9). HBsAg can be detected throughout the active disease process, and anti-HBs does not develop, which probably accounts for the chronicity of the disease. However, anti-HBc is detected. Two types of chronic hepatitis can be distinguished. In one, HBsAg is detected, but not HBeAg; these patients usually show progressive liver dysfunction. In the other, both antigens are found; development of antibody to HBeAg is associated with clinical improvement. Chronic infection with hepatitis B is best detected by persistence of HBsAg in blood for more than 6 to 12 months. Progression of liver disease is associated with more than 1000 IU of HBV DNA. Persons with levels lower than 1000 IU and normal liver function have a low risk of progression.

TREATMENT

There is no specific treatment recommended for acute hepatitis B. A high-calorie diet is desirable. Treatment should be considered for patients with rapid deterioration of liver function, cirrhosis or complications such as ascites, hepatic encephalopathy, or hemorrhage as well as those who are immunosuppressed. For chronic hepatitis B diseases, pegylated or regular interferon-α provides benefit in some patients. Lamivudine (3TC), a potent inhibitor of HIV reverse transcriptase, and other nucleoside analogs (entecavir, telbivudine) as well as certain nucleotide analogs (adfovir) are active against hepatitis B. These antivirals inhibit viral replication and may reduce viral load but do not cure HBV infection.

PREVENTION

Screening of blood and plasma product donors for HBsAg and anti-HBcAg has greatly reduced the incidence of hepatitis B in recipients. Similarly, screening pregnant women and treatment of exposed newborns with hepatitis B immune globulin (HBIG) and vaccine have reduced vertical transmission. Safe sexual practices and avoidance of needlestick injuries or injection drug use are approaches to diminishing the risk of hepatitis B infection. Both active prophylaxis and passive prophylaxis against hepatitis B infection can be accomplished. Most preparations of ISG contain only moderate levels of anti-HBs; however, specific HBIG with high titers of hepatitis B antibody is now available. HBIG is prepared from sera of subjects who have high titers of antibody to HBsAg, but are free of the antigen itself. Administration of HBIG soon after exposure to the virus greatly reduces the development of symptomatic disease. Postexposure prophylaxis with HBIG should be followed by active immunization with vaccine.

Purified inactivated HBsAg vaccine (subunit vaccine) from chronic carriers has been available for several years. This was developed by purification and inactivation of HBsAg from the blood of HBV-infected chronic carriers, but it is no longer in use. The current vaccine (ENGERIX-B, RECOMBIVAX-HB) is a recombinant product derived from HBsAg expressed in yeast. Excellent protection has been shown in studies of men who have sex with men and in medical personnel. These groups and others, such as laboratory workers, injection drug users, travelers to endemic areas, persons at risk for sexually transmitted diseases, and those in contact with patients who have chronic hepatitis B, should receive hepatitis B vaccine as the preferred method of preexposure prophylaxis. Recently, immunization of newborns, all children, and adolescents has been recommended. Three intramuscular doses (at 0, 1, and 6 months) are given to achieve maximum titer. Protection may not be lifelong.

Several combination vaccines are also available. These include COMVAX (hepatitis B-Haemophilus influenzae conjugate vaccine, cannot be given before 6 weeks or after 71 months), PEDIARIX (hepatitis B, diphtheria, tetanus, acellular pertussis, and inactivated polio, cannot be given before 6 weeks or after 7 years), and TWINRIX (hepatitis A and hepatitis B is recommended at the age of 18 years or above).

A combination of active and passive immunization is the most effective approach to prevent neonatal acquisition and chronic carriage in the neonate. Routine screening of pregnant women for the presence of HBsAg is recommended. Infants born to those who are positive should receive HBIG in the delivery room followed by three doses of hepatitis B vaccine beginning 24 hours after birth. A similar combination of passive and active immunization is used for unimmunized persons who have been exposed by needlestick or similar injuries. The procedure varies depending on the hepatitis B status of the “donor” case linked to the injury.

VIROLOGY

Delta hepatitis is caused by the hepatitis D virus (HDV). This small, single-stranded circular (–) RNA virus requires the presence of HBsAg (HBV surface antigen) for its transmission, and is thus found only in persons with acute or chronic HBV infection. Strategies directed at preventing HBV are also effective in preventing HDV. Associated with the circular RNA, which forms a rod because of extensive base pairing, are proteins of 27 and 29 kDa, which constitute the delta capsid antigen (HDV capsid antigen). This protein–RNA complex is surrounded by HBsAg (Figure 13–10). Thus, although the delta virus produces its own capsid antigens, it co-opts the HBsAg in assembling its coat or envelope. Unlike other RNA viruses, HDV genome is not capable of encoding its own RNA polymerase.

The replication of HDV involves virus entry in hepatocytes (liver cells) just like HBV, because HDV contains HBsAg on its surface. Because HDV lacks an RNA polymerase required for transcription and replication, it uses host cell RNA polymerase to synthesize mRNA and RNA genome in the nucleus. This is unique for an RNA virus to replicate in the nucleus without encoding its own RNA polymerase.

The extensive base pairing in some regions of the HDV genome allows the cellular RNA polymerase to bind the base-paired RNA sequences, as RNA polymerase binds to DNA sequences, and to transcribe HDV mRNA. The RNA genome further forms a ribozyme structure that allows self-cleaving of the RNA genome to generate mRNA. The delta capsid antigens are synthesized and associate with HDV circular RNA genomes followed by acquiring an envelope from endoplasmic reticulum or Golgi apparatus containing HBsAg. Thus, the presence of HBsAg is essential for assembly of HDV virions.

DELTA HEPATITIS DISEASE

Delta hepatitis is most prevalent in groups with a high risk for developing hepatitis B. Paradoxically, delta hepatitis is not common in East Asia, where hepatitis B is common, but it is most common in the Middle East, parts of Africa, and South America (Figure 13–11). Injection drug users are those at greatest risk in the western parts of the world, and up to 50% of such individuals may have IgG antibody to the delta virus antigen. Other risks include sexual transmission and dialysis. Vertical transmission can also occur.

CLINICAL ASPECTS

MANIFESTATIONS

Two major types of delta infection have been noted: Simultaneous delta and hepatitis B infections or delta superinfection in those with chronic hepatitis B. Simultaneous infection with both delta and hepatitis B may result in clinical hepatitis that is indistinguishable from acute hepatitis A or B, but it may manifest as a second rise in liver enzymes (AST, ALT). Persons with chronic hepatitis B who acquire superimposed infection with hepatitis D suffer relapses of jaundice and have a high likelihood of developing chronic cirrhosis. Epidemics of delta infection have occurred in populations with a high incidence of chronic hepatitis B and have resulted in rapidly progressive liver disease, causing death in up to 20% of infected persons.

DIAGNOSIS

Diagnosis of delta infection is made most commonly by demonstrating IgM or IgG antibodies, or both, to the delta capsid antigen in serum. The IgM antibodies appear within 3 weeks of infection and persist for several weeks, whereas IgG antibodies persist for years. In coinfection, the patient has both anti-HBc and anti-D antibodies, whereas in superinfection, the anti-HBc is already present and anti-D capsid antibodies appear later.

TREATMENT AND PREVENTION

Interferon and other anti-HBV therapies (nucleosides, nucleotide analogs) are not active against hepatitis D. Response to treatment in patients with delta hepatitis (and hepatitis B) is less than in those with hepatitis B alone.

Because the surface of delta hepatitis is HBsAg, measures aimed at limiting the transmission of hepatitis B (eg, vaccination, blood screening) prevent the transmission of delta hepatitis. Persons infected with hepatitis B or D should not donate blood, organ, tissues, or semen. Safe sex should be practiced unless there is only a single sex partner who is already infected. Methods of reducing transmission include decreased use of contaminated needles and syringes by injection drug users and use of needle safety devices by healthcare workers.

VIROLOGY

Hepatitis C virus (HCV) is an RNA-enveloped virus in the Flaviviridae family and Hepacivirus genus that is transmitted through blood and blood-derived products. Several other important members of Flaviviridae that cause disease in humans belong to Flavivirus genus, including yellow fever virus, dengue virus, West Nile virus that are arboviruses and transmitted through bite of arthropods (discussed in Chapter 16). HCV has a very simple, positive-sense, single-stranded RNA genome, consisting of just three structural (C, core; E1 and E2, envelope) and five nonstructural (NS2-NS5) genes. The HCV virion of 50 nm in diameter contains an RNA genome of 9.5 kb, which is enclosed in an icosahedral capsid or core (C) protein and a lipid-bilayer envelope containing two virus-specific glycoproteins E1 (gp31) and E2 (gp70) (Figure 13–12). The RNA genome is encoded into a polyprotein, which is processed into individual proteins by viral and host proteases. The envelope glycoproteins interact with receptor and coreceptor on the host cell for virus entry into target cells. In addition, antibodies against these envelope glycoproteins are involved in virus neutralization.

HCV is highly heterogeneous because the genome of HCV is highly mutable, because its RNA-dependent RNA polymerase lacks proofreading ability. Mutations give rise to HCV quasispecies (variants) and antigenic variation, most noticeably in the E2 glycoprotein hypervariable regions (HVR1 and HVR2), which may allow the virus to escape immune response and cause chronic or persistent infection in infected persons. The hypervariable region in E2 contains the epitope for neutralization, and mutations allow the newly generated HCV variants to escape preexisting immune response.

There are at least 11 genotypes, with multiple subtypes. The genotypes have different geographic distributions and may be associated with differing severity of disease as well as response to therapy. Genotypes 1-3 have worldwide distribution, with genotype 1a predominating in North America.

The HCV genome also encodes a nonstructural gene that is involved in sensitivity to interferon. HCV heterogeneity and generation of multiple HCV genotypes, like HIV, hinder the development of an HCV vaccine.

Similar to other positive-sense RNA viruses, HCV also replicates in the cytoplasm of the infected cell. Because of lack of a tissue culture system for HCV propagation, the replication cycle of HCV is not fully understood. In infected people, the virions of HCV are firmly associated with lipoproteins to form a complex particle called lipoviroparticle (LVP). These LVPs attach to heparan sulfate proteoglycans on the hepatocytes. HCV-LVPs may then interact with low-density lipoprotein receptor (LDLR) leading to a nonproductive infection. However, in the productive infection, HCV envelope glycoprotein (E2) interacts with scavenger receptor class B type I (SCARB1) and the tetraspanin CD81 (a member of the transmembrane 4 superfamily) followed by virus entry into target cells probably via receptor-mediated endocytosis. After virus entry, uncoating takes place followed by translation of a full-length genomic, positive-sense RNA via binding of ribosome to the internal ribosome entry site (IRES) located on viral RNA into a polyprotein, which is cleaved into individual proteins by proteases. One of these proteins is RNA-dependent RNA polymerase that directs transcription and replication via negative-sense RNA intermediates. Viral structural proteins (C, and E1 and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are synthesized as a polyprotein and then cleaved to mature proteins by viral (NS3/NS4A) and host proteases in the cytoplasm. Virus assembly takes place in the cytoplasm by formation of vesicles that fuses with the plasma membranes for virus release.

HEPATITIS C DISEASE

EPIDEMIOLOGY

Similar to HBV, HCV is spread parenterally. The transmission of HCV by blood was well documented. Indeed, until screening blood for transfusions was introduced, it caused most cases of posttransfusion hepatitis. Screening of donor blood for antibody has reduced posttransfusion hepatitis by 80% to 90%. HCV may be sexually transmitted but to a much lesser degree than HBV. Needle sharing accounts to up to 40% of the cases. Worldwide, 150 million are chronically infected with HCV and 3 to 4 million people are infected every year as well as 350 000 people die with HCV-related liver disease every year. The highest prevalence of HCV is in the Middle East, especially in Egypt. In the United States, 3.2 million people (1.1%) have antibody to hepatitis C and are chronically infected and more than 17 000 people are newly infected annually. Since the 1980s, outbreaks of hepatitis C have been associated with intravenous immune globulin (IVIG). To reduce this risk, all US-licensed IVIG products now have additional viral inactivation steps included in the manufacturing process. Furthermore, all immunoglobulin products (including intramuscular immunoglobulin products that have not been associated with hepatitis C) that lack viral inactivation steps are now excluded if HCV is detected by polymerase chain reaction (PCR). Other individuals considered at risk for hepatitis C are healthcare workers because of needlesticks and chronic hemodialysis patients and their spouses. Vertical transmission also occurs during deliveries.

PATHOGENESIS

HCV is transmitted via blood and blood-derived products and invades and infects the peripheral blood B and T lymphocytes and monocytes and moves to the main site of infection—the liver. The rate of HCV replication in hepatocytes is very high (ie, 1 × 10¹² virions per day), as 10% of the hepatic cells are infected. The high rate of viral replication results in an increased level of viral heterogeneity, which allows the virus to evade the host immune response. Although little evidence exists regarding a direct effect of HCV-induced cytopathic effects on the hepatocytes (liver cells), hepatocytes are likely killed by immune-mediated cytotoxic T cells. Several recent studies suggest that HCV replication can cause cytopathic lesions in the liver, such as histologic lesions with scant inflammatory infiltrate, and fulminant hepatitis C after chemotherapy in liver transplant recipients. The innate immune response results in the activation of cytokines and interferon, which initially control viral replication in some cases. However, HCV-encoded proteins help the virus to evade innate immune response, including interaction of HCV core with tumor necrosis factor (TNF) receptor, which decreases cytolytic T-cell activity and interference of HCV nonstructural (NS3/NS4A/NS4B) proteins with interferon pathways. In addition, the natural killer (NK) cells respond to HCV infection by releasing perforins, which fragment nuclei of infected cells and induce apoptosis. HCV infection is inhibited by the release of interferon-γ, which recruits intrahepatic inflammatory cells, stimulates helper T1 (T_H1) response, and induces necrosis or apoptosis of HCV-infected cells.

Adaptive immune responses, including cell-mediated and humoral responses are elicited after expression of HCV proteins, especially the envelope glycoproteins E1 and E2. HCV antibodies appear several weeks after infection, and because of selective pressure from the host, mutations take place in the E2/E1 proteins, allowing the virus to evade the humoral immune response and establish persistent infection. More important, HCV antibodies have been implicated in tissue damage because of immune complex formation. Examples of such tissue damage are antinuclear antibodies, autoantibodies that act against cytochrome P450, and antibodies that work against the liver and kidney.

The immune complexes are also deposited in other tissues and cause some of the other extrahepatic problems, including vasculitis, arthritis, glomerulonephritis, and others. In the absence of strong humoral immune response against HCV infection, CTLs or CD8 T cells are critical to the elimination of HCV infection, and any impairment in cell-mediated immunity could be a major factor for a high level of chronicity in infected patients. The CD8 T cells eliminate HCV by apoptosis of infected hepatocytes and interferon-γ-induced inhibition of viral replication. The CTL response is less effective in chronically HCV-infected patients compared with that in acutely infected patients. Also, CD4 T cells play an important role in HCV pathogenesis by secreting several proinflammatory cytokines related to hepatocyte death. During acute infection, the rise in serum transaminases corresponds with cell damage, and the hepatic lesion is immune mediated. The chronic infection probably progresses as a result of imbalance between T_H1 and T_H2 cytokines. T_H1 cytokines such as interleukin 2 (IL-2) and TNF-α are associated with aggressive hepatic disease, whereas T_H2 cytokines (IL-10) are related to the milder presentation. Expression of TNF-α causes hepatic injury and triggers “cytokine storm” to cause liver damage in chronically infected patients (Figure 13–13). Chronic HCV infection promotes insulin resistance in hepatocytes by increasing the inflammatory response due to increased expression of TNF-α and IL-6 and oxidative stress. Insulin resistance may lead to the progression of fibrosis and hepatocarcinogenesis.

In addition to immune status of the host, genetic host factors play an important role in HCV pathogenesis. One such factor is major histocompatibility complex (MHC) class II DR5 allele, which has been shown to be associated with a lower incidence of cirrhosis in HCV-infected individuals. One study identified CTLs restricted by HLA A2 in 97% of chronic hepatitis C patients. Several extrinsic factors, such as alcohol abuse and smoking, are related to progression of chronic hepatitis C. The influence of age, gender, and race due to genetic factor variation has been implicated with progression of hepatitis C. Coinfection with other viruses such as HIV, HBV, HAV, and human T-lymphotropic virus influence the outcome of HCV disease.

HCV-infected patients may develop cirrhosis of liver with increased risk of HCC. It has also been suggested that alcoholism increases the rate of HCC in HCV-infected patients. It is also believed that HCC is probably caused by long-term damage followed by rapid growth rate of hepatocytes during regeneration of liver, which may be mediated by some cytokines. Recent studies suggest that various HCV protein–host-cell interactions may play a role in the development of HCC, including disturbance in the cell cycle, upregulation of oncogenes, and loss of tumor suppressor gene functions. HCV core protein has been shown to perturb and modify the growth of the cell cycle. HCV core interacts directly or indirectly with components or pathways that lead to oncogenesis such as tumor suppressor genes (p53, p73), protein kinase, cell cycle, and cell proliferation and differentiation. In addition, HCV nonstructural proteins, NS3 and NS5A, and HCV core protein play a role in cell transformation, differentiation, and oncogenesis.

CLINICAL ASPECTS

MANIFESTATIONS

The incubation period of hepatitis C averages 6 to 12 weeks. The infection is usually asymptomatic or mild and anicteric in 75%, but it results in a chronic carrier state in up to 85% of adult patients. Fulminant hepatitis due to hepatitis C is very rare in the United States. The average duration of time from infection to the development of chronic hepatitis is 10 to 18 years. Cirrhosis and HCC are late sequelae of chronic hepatitis. Chronic hepatitis tends to wax and wane, is often asymptomatic, and may be associated with either elevated or normal ALT values in serum (Figure 13–14). Chronic hepatitis C is the leading infectious cause of chronic liver disease and liver transplantation in the United States.

DIAGNOSIS

HCV antigens are not detectable in blood, so diagnostic tests attempt to demonstrate the presence of HCV antibodies. Unfortunately, the antibody responses in acute disease remain negative for 1 to 3 weeks after clinical onset and may never become positive in up to 20% of patients with acute, resolving disease. Current tests detect antibodies to multiple hepatitis C antigens first by enzyme-linked immunosorbent assay (ELISA) and then confirmed by recombinant immune immunoblot assay (RIBA). Whereas in ELISA the positive results demonstrate the presence of HCV antibodies, the RIBA test identifies antibodies against specific HCV antigens (core, NS3, and NS5). Even with these newer assays, IgG antibody to hepatitis C may not develop for up to 4 months, making the serodiagnosis of acute hepatitis C difficult. Quantitative assays of hepatitis C RNA by RT-PCR may be used for diagnosis, predicting IFN responsiveness and monitoring therapy, but there is not a very good correlation between viral load and histology. Genotyping is important for therapy, since type 1 (the most common in the United States) requires the longest period of therapy. A next-generation HCV RNA test has been approved by the FDA that would accurately quantitate HCV RNA in patients to assess the response to antiviral agents.

TREATMENT AND PREVENTION

Combination treatment of acute hepatitis C appears beneficial, but can be deferred for weeks to determine whether the infection resolves spontaneously. Combination therapy with interferon-α and ribavirin is the current treatment of choice for patients with evidence of chronic hepatitis due to HCV. Interferon-α is available in the form of injection and ribavirin as an oral pill. Criteria for initiating treatment are controversial, but most physicians would initiate treatment in a patient with abnormal liver histology and elevated liver enzymes. Responses are better in patients with genotypes other than 1 and those with low initial titers of viral RNA. The duration of treatment depends on HCV genotypes and viral load in patients. HCV-infected patients with genotype 2 or 3 are given the combination treatment for 24 weeks, whereas patients infected with genotype 1 require treatment for 48 weeks. In 2011, FDA approved two HCV protease (NS3/4A) inhibitors, Victrelis (boceprevir) and Incivek (telaprevir), to be used in combination of interferon-α and ribavirin in patients infected with HCV genotype 1 who have not been treated before or failed with previous treatment. HCV protease inhibitors, if given alone, generate virus resistance quickly. Side effects of telaprevir include anemia, rash, nausea, diarrhea, headache, and rectal irritation and pain.

Corticosteroids are not beneficial. Avoidance of injection drug use and screening of blood products are important preventive measures. Prophylactic ISG does not protect against hepatitis C.

VIROLOGY

Hepatitis E virus (HEV) is the cause of another form of hepatitis that is spread by the fecal–oral route, and therefore resembles hepatitis A. It used to be referred as enterically transmitted (ET) non-A, non-B hepatitis. HEV is a positive-sense, single-stranded RNA virus that is similar to, but distinct from, caliciviruses. The viral particles in stool are naked capsid, spherical, 27 to 34 nm in diameter with icosahedral symmetry, and unenveloped, and they exhibit spikes on their surface. The genome of HEV is 7.2 kb in size and contains three open reading frames (ORFs). ORF-1 encodes the nonstructural proteins, including methyltransferase, protease, helicase, and RNA-dependent RNA polymerase. ORF-2 encodes capsid protein and ORF-3 a multifunctional small protein.

Like other positive-sense RNA viruses, HEV replicates in the cytoplasm. HEV enters host cells via an unidentified receptor. After uncoating, the positive-sense RNA genome is released in the cytoplasm that acts as an mRNA for synthesis of ORF-1 (nonstructural proteins). The viral RNA-dependent RNA polymerase transcribes a replicative intermediate negative-sense RNA that serves template for a subgenomic RNA and full-length genomic RNA. The subgenomic RNA synthesizes ORF-2 (capsid) and ORF-3. Virus assembly takes place in the cytoplasm and ORF-3 helps virus release from the infected cells.

EPIDEMIOLOGY

Twenty million HEV infections occur every year worldwide, including more than 3 million acute cases and approximately 70 000 deaths. Most cases of hepatitis E infection have been identified in developing countries with poor sanitation (eg, Asia, Africa, and the Indian subcontinent), and recurrent epidemics have been described in these areas (Figure 13–15). Cases have been recently recognized in developed countries such as the United States; most have been in visitors or immigrants from endemic areas.

CLINICAL ASPECTS

HEV is transmitted fecally–orally, mainly from contaminated drinking water. Several other transmission routes been documented, including foodborne transmission from ingestion of infected animal products, zoonotic transmission from animals to humans, transfusion of infected blood products and vertical transmission. Whereas major outbreaks are caused by contaminated water or food supplies, sporadic outbreaks occur from ingesting raw or uncooked shellfish. Similar to hepatitis A, infection with this virus is frequently subclinical. The incubation period for hepatitis E is approximately 40 days. When symptomatic, the resulting acute disease may be fatal, especially in pregnant women. In endemic, developing areas, hepatitis E has the highest attack rate in young adults aged 15 to 40 years. It does not appear to spread from person to person. Symptoms of HEV include jaundice, loss of appetite, enlarged liver, nausea and vomiting, and fever that last for 1 to 2 weeks.

DIAGNOSIS

Because HEV is clinically indistinguishable from other acute hepatitis, the diagnosis is confirmed by demonstrating the presence of specific IgM antibody, although very few laboratories perform this test. HEV RNA may be detected by RT-PCR, although this test is used mainly for research purposes.

TREATMENT AND PREVENTION

There is no specific treatment available, other than supportive measures and proper nutrition. ISG does not appear to provide protection. The risk of transmission can be reduced by upholding safe hygienic practices, drinking safe and boiled water, and avoiding eating raw and uncooked seafood, vegetables, and fruits in endemic areas. In seriously ill patients with liver failure, liver transplantation may be the only recourse.

In 1995, hepatitis G virus (HGV), or GB virus C (GBV-C), was discovered in sera from two patients. HGV and GBV-C are two isolates of the same virus. Hepatitis G is a (+) sense RNA virus of 9.3 kb, similar to that of hepatitis C and members of the Flaviviridae family, but has not been associated with any clinical disease. The virion structure of hepatitis G is similar to that of HCV. The genome encodes two structural envelope proteins (E1 and E2) and five nonstructural proteins (NS2, NS3, NS4b, NS5a, and NS4b). The other structural protein, core or capsid, has not been characterized. The virus encodes its own RNA-dependent RNA polymerase. HGV has been found to replicate in lymphocytes rather than in hepatocytes. HGV is mainly transmitted parenterally, including blood and blood-derived products. There may be a sexual component of transmission as seen in HBV and HCV. HGV infection is widely distributed worldwide with a high prevalence in blood donors in the United States. Approximately 10% to 30% of the blood donors have antibody against HGV. An antibody assay can detect past, but not present, infection, and detection of acute infection with hepatitis G requires a PCR assay for viral RNA in serum. Up to 5% of volunteer blood donors and 35% of HIV-infected patients are positive for hepatitis G RNA. In addition to being closely related to hepatitis C, data suggest that 10% to 20% of patients infected with hepatitis C are also infected with hepatitis G. Given this association, it has been difficult to ascertain the contribution of hepatitis G to clinical disease. Patients infected with both viruses (HCV and HGV) do not appear to have worse disease than those infected by HCV only. Currently, there is no useful serologic test and no therapy is established for patients with HGV.

Recent studies suggest that persistent coinfection of HGV and HIV is associated with lower viral (HIV) load, higher CD4⁺ T-cell count and prolonged survival of HIV-infected individuals. Some studies suggest that HGV E2 protein inhibits processing of HIV Gag precursor protein resulting in inhibition of virus assembly and release. Other studies suggest that HGV stimulates cytokine production that inhibits HIV replication, decreases T-cell activation and proliferation, and downregulates chemokine receptors, CCR5 and CXCR4 (HIV coreceptors). However, more research is needed before any of these findings is translated into therapeutic advances.