Hepatitis B Virus hepadnaviruses genome is a small, circular, partly double-stranded DNA

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1 Hepatitis B Virus HBV is the major member of the hepadnaviruses. Other members of this family (Box 65-3) include woodchuck, ground squirrel, and duck hepatitis viruses. These viruses have limited tissue tropisms and host ranges. HBV infects the liver and to a lesser extent the kidneys and pancreas of only humans and chimpanzees. Advances in molecular biology have made it possible to study HBV, despite the limited host range of the virus and difficult cell-culture systems in which to grow it. Structure HBV is a small, enveloped DNA virus with several unusual properties (Figure 65-4). Specifically, the genome is a small, circular, partly double-stranded DNA of only 3200 bases. Although a DNA virus, it encodes a reverse transcriptase and replicates through an RNA intermediate. Box Unique Features of Hepadnaviruses HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus. Virus has enveloped virion containing partially doublestranded, circular DNA genome. Replication is through a circular RNA intermediate. Virus encodes and carries a reverse transcriptase. Virus encodes several proteins (HBsAg [L, M, S]; HBe/HBc) that share genetic sequences but with different in-frame start codons. HBV has a strict tissue tropism to the liver. HBV-infected cells produce and release large amounts of HBsAg particles lacking DNA. The HBV genome can integrate into the host chromosome. page 648 page 649 Figure 65-4 Hepatitis B virus (Dane particle) and hepatitis B surface antigen (HBsAg) particles. The spherical HBsAg consists mainly of the S form of HBsAg, with some M. The fiber HBsAg has S, M, and L forms. Bp, base pair; L, gp42; M, gp36; S, gp27. The virion, also called the Dane particle, is 42 nm in diameter. The virions are unusually stable for an enveloped virus. They resist treatment with ether, low ph, freezing, and moderate heating. These characteristics assist transmission from one person to another and hamper disinfection. The HBV virion includes a protein kinase and a polymerase with reverse transcriptase and ribonuclease H activity, as well as a P protein attached to the genome. All of this is surrounded by an icosahedral capsid formed by the hepatitis B core antigen (HBcAg) and an envelope containing three forms of the glycoprotein

2 hepatitis B surface antigen (HBsAg). A hepatitis Be antigen (HBeAg) protein shares most of its protein sequence with HBcAg but is processed differently by the cell, is primarily secreted into serum, does not self-assemble (like a capsid antigen), and expresses different antigenic determinants. HBsAg-containing particles are released into the serum of infected people and outnumber the actual virions. These particles can be spherical (but smaller than the Dane particle) or filamentous (see Figure 65-4). They are immunogenic and were processed into the first commercial vaccine against HBV. HBsAg, originally termed the Australia antigen, includes three glycoproteins (L, M, and S) encoded by the same gene and read in the same frame but translated into protein from different AUG start codons. The S (gp27; 24 to 27 kda) glycoprotein is completely contained in the M (gp36; 33 to 36 kda) glycoprotein, which is contained in the L (gp42; 39 to 42 kda) glycoprotein; all share the same C-terminal amino acid sequences. All three forms of HBsAg are found in the virion. The S glycoprotein is the major component of HBsAg particles; it self-associates into 22-nm spherical particles that are released from the cells. The filamentous particles of HBsAg found in serum contain mostly S but also small amounts of the M and L glycoproteins and other proteins and lipids. In one orientation, the L glycoprotein binds the virus to receptors on liver cells, and in another orientation, it binds the envelope to the capsid to assemble the virion. The glycoproteins of HBsAg contain the group-specific (termed a) and type-specific determinants of HBV (termed d or y and w or r). Combinations of these antigens (e.g., ady and adw) result in eight subtypes of HBV that are useful epidemiologic markers. Replication The replication of HBV is unique for several reasons (see Box 65-1). First, HBV has a distinctly defined tropism for the liver. Its small genome also necessitates economy, as illustrated by the pattern of its transcription and translation. In addition, HBV replicates through an RNA intermediate and produces and releases antigenic decoy particles (HBsAg) (Figure 65-5). The attachment of HBV to hepatocytes is mediated by the HBsAg glycoproteins. Several liver cell receptors have been suggested, including the transferrin receptor, the asialoglycoprotein receptor, and human liver annexin V. The mechanism of entry is not known, but HBsAg binds to polymerized human serum albumin and other serum proteins, and this interaction may facilitate binding and uptake of the virus by the liver. page 649 page 650 Figure 65-5 Replication of hepatitis B virus. After entry into the hepatocyte and uncoating of the nucleocapsid core, the partially double-stranded DNA genome is completed by enzymes in the core and then delivered to the nucleus. Transcription of the

3 genome produces four messenger RNAs (mrnas), including an mrna larger than the genome (3500 bases). The mrna then moves to the cytoplasm and is translated into protein. Core proteins assemble around the 3500-base mrna, and negativesense DNA is synthesized by a reverse transcriptase activity in the core. The RNA is then degraded as a positive-sense (+) DNA is synthesized. The core is enveloped before completion of the positive-sense DNA and then released by exocytosis. On penetration into the cell, the partial DNA strand of the genome is completed by being formed into a complete double-stranded DNA circle, and the genome is delivered to the nucleus. Transcription of the genome is controlled by cellular transcription elements found in hepatocytes. The DNA is transcribed from different starting points on the circle but have the same 3' end. There are three major classes (2100, 2400, and 3500 bases) and two minor classes (900 bases) of overlapping messenger RNAs (mrnas) (Figure 65-6). The 3500-base mrna is larger than the genome. It encodes the HBc and HBe antigens, the polymerase, and a protein primer for DNA replication and acts as the template for replication of the genome. The HBe and HBc are related proteins that are translated from different in-phase start codons of closely related mrna. This causes differences in their processing and structure, with shedding of the HBe and incorporation of HBc into the virion. Similarly, the base mrna encodes the small and medium glycoproteins from different in-phase start codons. The 2400-base mrna, which encodes the large glycoprotein, overlaps the 2100-base mrna. The 900-base mrna encodes the X protein, which promotes viral replication as a transactivator of transcription and as a protein kinase. Figure 65-6 DNA, RNA, mrna, and proteins of hepatitis B virus (HBV). The inner green circles represent the DNA genome with the nucleotide number at the center. DR1 and DR2 are direct repeat sequences of DNA and are important for replication and integration of the genome. The 3500-base transcript (outer black thin-line circle) is larger than the genome and is the template for replication of the genome. Bold arcs represent mrna for viral proteins. Note that several proteins are translated from the same mrna but from different AUG codons and that different mrnas overlap. AAA, 3' polya at end of mrn; C, C mrna (HBcAg); E, E mrna (HBeAg); l, large glycoprotein; m, medium glycoprotein; P, polymerase-protein primer for replication; s, small glycoprotein; S, S mrna (HBsAg); X, X mrna. (Modified from Armstrong D, Cohen J: Infectious Diseases. St Louis, Mosby, 1999.) Replication of the genome utilizes the larger-than-genome, 3500-base mrna. This is packaged into the core nucleocapsid that contains the RNA-dependent DNA polymerase (P protein). This polymerase has reverse transcriptase and ribonuclease H activity but HBV lacks the integrase activity of the retroviruses. The 3500-base RNA acts as a template, and negative-strand DNA is synthesized using a protein primer from the P protein, which remains covalently attached to the 5' end. After this, the RNA is degraded by the ribonuclease H activity as the positive-strand DNA is synthesized from the negative-sense DNA template. However, this process is interrupted by envelopment of the nucleocapsid at HBsAg-containing intracellular membranes, thereby capturing genomes containing RNA-DNA circles with different lengths of RNA. Continued degradation of the remainder of the RNA in the virion yields a partly double-stranded DNA genome. The virion is then released from the hepatocyte by exocytosis without killing the cell, not by cell lysis. The entire genome can also be integrated into the host cell chromatin. HBsAg, but not other proteins, can often be detected in the cytoplasm of cells containing

4 integrated HBV DNA. The significance of the integrated DNA in the replication of the virus is not known, but integrated viral DNA has been found in hepatocellular carcinomas. page 650 page 651 Pathogenesis and Immunity HBV can cause acute or chronic, symptomatic or asymptomatic disease. Which of these occurs seems to be determined by the person's immune response to the infection (Figure 65-7). Detection of both the HBsAg and the HBeAg components of the virion in the blood indicates the existence of an ongoing active infection. HBsAg particles continue to be released into the blood, even after virion release has ended and until the infection is resolved. The major source of infectious virus is blood, but HBV can be found in semen, saliva, milk, vaginal and menstrual secretions, and amniotic fluid. The most efficient way to acquire HBV is through injection of the virus into the bloodstream (Figure 65-8). Common but less efficient routes of infection are sexual contact and birth. Figure 65-7 Major determinants of acute and chronic hepatitis B virus (HBV) infection. HBV infects the liver but does not cause direct cytopathology. Cell-mediated immune lysis of infected cells produces the symptoms and resolves the infection. Insufficient immunity can lead to chronic disease. Chronic HBV disease predisposes a person to more serious outcomes. Purple arrows indicate symptoms; green arrows indicate a possible outcome. Figure 65-8 Spread of hepatitis B virus (HBV) in the body. Initial infection with HBV occurs through injection, heterosexual and homosexual sex, and birth. The virus then spreads to the liver, replicates, induces a viremia, and is transmitted in various body secretions in addition to blood to start the cycle again. Symptoms are caused by cell-mediated immunity (CMI) and immune complexes between antibody and hepatitis B surface antigen (HBsAg). IV, intravenous. The virus starts to replicate in the liver within 3 days of its acquisition, but as already noted, symptoms may not be observed for 45 days or longer, depending on the infectious dose, the route of infection, and the person. The virus replicates in hepatocytes with minimal cytopathic effect. Infection proceeds for a relatively long time without causing liver damage (i.e., elevation of liver enzyme levels) or symptoms. During this time, copies of the HBV genome integrate into the hepatocyte chromatin and remain latent. Intracellular buildup of filamentous forms of HBsAg can produce the ground-glass hepatocyte cytopathology characteristic of HBV infection. Cell-mediated immunity and inflammation are responsible for causing the symptoms and effecting resolution of the HBV infection by eliminating the infected hepatocyte.

5 Epitopes from the HBc antigen are prominent T-cell antigens. An insufficient T-cell response to the infection generally results in the occurrence of mild symptoms, an inability to resolve the infection, and the development of chronic hepatitis (see Figure 65-7). Antibody (as generated by vaccination) can protect against initial infection by preventing delivery of the virus to the liver. Later in the infection, the large amount of HBsAg in serum binds to and blocks the action of neutralizing antibody, which limits the antibody's capacity to resolve an infection. Immune complexes formed between HBsAg and anti-hbs contribute to the development of hypersensitivity reactions (type III), leading to problems such as vasculitis, arthralgia, rash, and renal damage. Infants and young children have an immature cell-mediated immune response and are less able to resolve the infection, but they suffer less tissue damage and milder symptoms. As many as 90% of infants infected perinatally become chronic carriers. Viral replication persists in these people for long periods. page 651 page 652 During the acute phase of infection, the liver parenchyma shows degenerative changes consisting of cellular swelling and necrosis, especially in hepatocytes surrounding the central vein of a hepatic lobule. The inflammatory cell infiltrate is mainly composed of lymphocytes. Resolution of the infection allows the parenchyma to regenerate. Fulminant infections, activation of chronic infections, or coinfection with the delta agent can lead to permanent liver damage and cirrhosis. Epidemiology In the United States, more than 12 million people have been infected with HBV (1 out of 20), with 5000 deaths per year. In the world, one out of three people have been infected with HBV, with approximately a million deaths per year. In developing nations, as much as 15% of the population may be infected during birth or childhood. High rates of seropositivity are observed in Italy, Greece, Africa, and Southeast Asia (Figure 65-9). In some areas of the world (southern Africa and southeastern Asia), the seroconversion rate is as high as 50%. PHC, a long-term sequela of the infection, is also endemic in these regions. The many asymptomatic chronic carriers with virus in blood and other body secretions foster the spread of the virus. In the United States, 0.1% to 0.5% of the general population are chronic carriers, but this is very low in comparison with many areas of the world. Carrier status may be lifelong. Figure 65-9 Worldwide prevalence of hepatitis B carriers and primary hepatocellular carcinoma. (Courtesy of Centers for Disease Control and Prevention, Atlanta.) Box High-Risk Groups for Hepatitis B Virus Infection People from endemic regions (i.e., China, parts of Africa, Alaska, Pacific Islands)

6 Babies of mothers with chronic hepatitis B virus Intravenous drug abusers People with multiple sex partners, homosexual and heterosexual Hemophiliacs and other patients requiring blood and blood product treatments Health care personnel who have contact with blood Residents and staff members of institutions for the mentally retarded Hemodialysis patients and blood and organ recipients The virus is spread by sexual, parenteral, and perinatal routes. Transmission occurs through contaminated blood and blood components by transfusion, needle sharing, acupuncture, ear piercing, or tattooing and through very close personal contact involving the exchange of semen, saliva, and vaginal secretions (e.g., sex, childbirth) (see Figure 65-8). Medical personnel are at risk in accidents involving needle sticks or sharp instruments. People at particular risk are listed in Box Sexual promiscuity and drug abuse are major risk factors for HBV infection. HBV can be transmitted to babies through contact with the mother's blood at birth and in the mother's milk. Babies born to chronic HBV-positive mothers are at highest risk for infection. Serologic screening of donor units in blood banks has greatly reduced the risk of acquisition of the virus from contaminated blood or blood products. Safer sex habits adopted to prevent HIV transmission and the administration of the HBV vaccine have also been responsible for decreasing the transmission of HBV. One of the major concerns about HBV is its association with PHC. This type of carcinoma probably accounts for 250,000 to 1 million deaths per year worldwide; in the United States, approximately 5000 deaths per year are attributed to PHC. Clinical Syndromes Acute Infection As already noted, the clinical presentation of HBV in children is less severe than that in adults, and infection may even be asymptomatic. Clinically apparent illness occurs in as many as 25% of those infected with HBV (Figures to 65-12). page 652 page 653 Figure Symptoms of typical acute viral hepatitis B infection are correlated with the four clinical periods of this disease. RUQ, right upper quadrant. (Redrawn from Hoofnagle JH: Lab Med 14: , 1983.)

7 HBV infection is characterized by a long incubation period and an insidious onset. Symptoms during the prodromal period may include fever, malaise, and anorexia, followed by nausea, vomiting, abdominal discomfort, and chills. The classic icteric symptoms of liver damage (e.g., jaundice, dark urine, pale stools) follow soon thereafter. Recovery is indicated by a decline in the fever and renewed appetite. Fulminant hepatitis occurs in approximately 1% of icteric patients and may be fatal. It is marked by more severe symptoms and indications of severe liver damage, such as ascites and bleeding. Figure Clinical outcomes of acute hepatitis B infection. (Redrawn from White DO, Fenner F: Medical Virology, 3rd ed. New York, Academic, 1986.) Figure A, The serologic events associated with the typical course of acute hepatitis B disease. B, Development of the chronic hepatitis B virus carrier state. Routine serodiagnosis is difficult during the hepatitis B surface antigen (HBsAg) window, when HBs and anti-hbs are undetectable. Anti-HBs, antibody to HBsAg; anti-hbc, antibody to HBcAg; anti-hbe, antibody to HBeAg. (Redrawn from Hoofnagle JH: Annu Rev Med 32:1-11, 1981.) HBV infection can promote hypersensitivity reactions that are caused by immune complexes of HBsAg and antibody. These may produce rash, polyarthritis, fever, acute necrotizing vasculitis, and glomerulonephritis. Chronic Infection Chronic hepatitis occurs in 5% to 10% of people with HBV infections, usually after mild or inapparent initial disease. Approximately one third of these people have chronic active hepatitis with continued destruction of the liver leading to scarring of the liver, cirrhosis, liver failure, or PHC. The other two thirds have chronic passive hepatitis and are less likely to have problems. Chronic hepatitis may be detected accidentally by finding elevated liver enzyme levels on a routine blood chemistry profile. Chronically infected people are the major source for spread of the virus and are at risk for fulminant disease if they become coinfected with HDV. Primary Hepatocellular Carcinoma page 653 page 654 The World Health Organization estimates that 80% of all cases of PHC can be attributed to chronic HBV infections. The HBV genome is integrated into these PHC cells, and the cells express HBV antigens. PHC is usually fatal and is one of the

8 three most common causes of cancer mortality in the world. In Taiwan, at least 15% of the population is carriers of HBV, and nearly half die of PHC or cirrhosis. PHC may become the first vaccine-preventable human cancer. HBV may induce PHC by promoting continued liver repair and cell growth in response to tissue damage or by integrating into the host chromosome and stimulating cell growth directly. Such integration could stimulate genetic rearrangements or juxtapose viral promoters next to cellular growth-controlling genes. Alternatively, a protein encoded by the HBV X gene may transactivate (turn on) the transcription of cellular proteins and stimulate cell growth. The presence of the HBV genome may allow a subsequent mutation to promote carcinogenesis. The latency period between HBV infection and PHC may be as short as 9 years or as long as 35 years. Laboratory Diagnosis The initial diagnosis of hepatitis can be made on the basis of the clinical symptoms and the presence of liver enzymes in the blood (see Figure 65-12). However, the serology of HBV infection describes the course and the nature of the disease (Table 65-2). Acute and chronic HBV infections can be distinguished by the presence of HBsAg and HBeAg in the serum and the pattern of antibodies to the individual HBV antigens. HBsAg and HBeAg are secreted into the blood during viral replication. The detection of HBeAg is the best correlate to the presence of infectious virus. A chronic infection can be distinguished by the continued finding of HBeAg, HBsAg, or both, and a lack of detectable antibody to these antigens. Table Interpretation of Serologic Markers of Hepatitis B Virus Infection Disease State Healthy State Serologic Early Early Late Reactivity (Presymptomatic) Acute Acute Chronic Acute Resolved Vaccinated Anti-HBc - - -* + +/- + - Anti-HBe /- +/- - Anti-HBs HBeAg HBsAg Infectious virus *Anti-HBc IgM should be present. Anti-HBe may be negative after chronic disease. HBeAg, hepatitis Be antigen; HBsAg, hepatitis B surface antigen. During the symptomatic phase of infection, detection of antibodies to HBeAg and HBsAg is obscured because the antibody is complexed with antigen in the serum.

9 The best way to diagnose a recent acute infection, especially during the period when neither HBsAg nor anti-hbs can be detected (the window), is to measure IgM anti- HBc. Treatment, Prevention, and Control Although no treatment is available for acute infection, Hepatitis B immune globulin may be administered within a week of exposure and to newborn infants of HBsAgpositive mothers to prevent and ameliorate disease. Chronic HBV infection can be treated with drugs targeted at the polymerase-for example, lamivudine (2'3'dideoxy- 3'-thiacytidine), which is also an HIV (human immunodeficiency virus) reverse transcriptase inhibitor-or the nucleoside analogues, adefovir dipivoxil and famciclovir. These FDA-approved treatments are taken for 1 year. Interferon-α (IFN-α) can also be effective and is taken for at least 4 months. Transmission of HBV in blood or blood products has been greatly reduced by screening donated blood for the presence of HBsAg and anti-hbc. Additional efforts to prevent transmission of HBV consist of avoiding sex with a carrier of HBV and avoiding the lifestyles that facilitate spread of the virus. Household contacts and sexual partners of HBV carriers are at increased risk, as are patients undergoing hemodialysis, recipients of pooled plasma products, health care workers exposed to blood, and babies born of HBV-carrier mothers. Vaccination is recommended for infants, children, and especially people in high-risk groups (see Box 65-4). For newborns of HBsAg-positive mothers and people accidentally exposed either percutaneously or permucosally to blood or secretions from an HBsAg-positive person, vaccination is useful even after exposure. Immunization of mothers should decrease the incidence of transmission to babies and older children, also reducing the number of chronic HBV carriers. Prevention of chronic HBV will reduce the incidence of PHC. page 654 page 655 The HBV vaccines are subunit vaccines. The initial HBV vaccine was derived from the 22-nm HBsAg particles in human plasma obtained from chronically infected people. The current vaccine was genetically engineered and is produced by the insertion of a plasmid containing the S gene for HBsAg into a yeast, Saccharomyces cerevisiae. The protein self-assembles into particles, which enhances its immunogenicity. The vaccine must be given in a series of three injections, with the second and third given 1 and 6 months after the first. More than 95% of individuals receiving the full three-dose course will develop protective antibody. The single serotype and limited host range (humans) help ensure the success of an immunization program. Universal blood and body fluid precautions are used to limit exposure to HBV. It is assumed that all patients are infected. Gloves are required for handling blood and body fluids; wearing protective clothing and eye protection may also be necessary. Special care should be taken with needles and sharp instruments. HBVcontaminated materials can be disinfected with 10% bleach solutions, but unlike most enveloped viruses, HBV is not readily inactivated by detergents.

10 Printed from STUDENT CONSULT: Medical Microbiology 6E (on 17 March 2010) 2010 Elsevier

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