The natural history of chronic hepatitis B virus infection

Three stages of chronic hepatitis B virus (HBV) infection are recognized: the immune tolerant phase, the chronic hepatitis B phase, and the inactive hepatitis B carrier phase. Active liver disease is most often found in persons with elevated aminotransferase levels and HBV DNA levels >10(5) copies/mL. Possible risk factors for developing liver disease include older age, male gender, presence of hepatitis B e antigen (HBeAg), HBV genotype, mutations in the precore and core promoter regions of the viral genome, and coinfection with hepatitis D (delta) virus. All persons chronically infected with HBV should be followed every 6 to 12 months with aminotransferase levels. Those with elevated levels should be tested for HBeAg and its antibody (anti-HBe) as well as HBV DNA levels to determine if they are in need of further evaluation with a liver biopsy and are candidates for antiviral therapy. Future research will help clarify the outcome of chronic HBV infection.     

Hepatitis B virus and hepatitis D virus replication in HBsAg-positive fulminant hepatitis

Hepatitis B virus DNA and hepatitis D virus RNA, the most sensitive markers of hepatitis B and hepatitis D virus replication, were sought by molecular hybridization with radioactive probes in serial serum samples from 29 consecutive patients with HBsAg-positive fulminant hepatitis. Nineteen patients had evidence of hepatitis D virus infection, as assessed by the presence in serum of delta antigen, anti-delta antibodies, or both. Hepatitis B virus DNA was found in only two patients: one was a chronic HBsAg carrier with hepatitis D virus superinfection and the other had fulminant hepatitis caused by hepatitis B and hepatitis D coinfection. Hepatitis D virus RNA was detected in three patients: two with hepatitis B and hepatitis D coinfection and also in the HBsAg carrier with positive hepatitis B virus DNA and hepatitis D virus superinfection. None of 10 patients with hepatitis B virus infection alone had detectable viral nucleic acids in serum. Overall, viral nucleic acids were detected in the sera of 4 of the 29 patients (14%). Hepatitis D virus antigenemia did not indicate hepatitis D virus replication because hepatitis D virus RNA was not detected in 9 of 12 patients with hepatitis D virus antigen in their sera. The low frequency of viral replication found in fulminant hepatitis B or D may explain the low recurrence rate of viral hepatitis in patients with fulminant hepatitis who have received liver transplantations.     

Precore mutant hepatitis B virus infection and liver disease.

The type of hepatitis B virus ("wild-type" or precore mutant) in anti-e antigen antibody-positive carriers, viral DNA levels in the serum, and core and e antigen expression in the liver were investigated to search for a possible correlation of these factors with the severity of liver damage. Two major groups of patients were found: the patients in group A were predominantly infected with precore mutant virus and had chronic active hepatitis, expressed nuclear/cytoplasmic core and e antigens in liver biopsy specimens, and usually had high levels of viral DNA in their serum; patients in group B were infected with a mixture of wild-type and mutant viruses, had predominantly chronic persistent hepatitis, showed weaker expression of nuclear core antigen with no cytoplasmic core or e antigen, and had low viremia. A few patients were infected with viruses without precore stop-codon mutation. These data indicate a high prevalence of precore mutant viruses in anti-e carriers with chronic liver disease and suggest that monitoring of virus sequence type and DNA level may be of prognostic value for liver disease sequelae.     

Long-term follow-up of anti-HBe-positive chronic active hepatitis B

Twenty-eight patients with chronic active hepatitis without cirrhosis who were positive for hepatitis B surface antigen and antibody to hepatitis B e antigen were followed for 1 to 15 years (mean 6.6 years) and underwent follow-up biopsy. At presentation, 12 of the 28 patients (43%) had hepatitis B virus DNA in serum, 10 (36%) had serologic evidence of hepatitis delta virus infection and 6 (21%) had no serologic markers of either hepatitis B virus replication or hepatitis delta virus infection. During follow-up, 15 (54%) patients developed active cirrhosis, including eight patients with hepatitis delta virus infection and five with hepatitis B virus DNA in serum. In seven (47%) of the 15 patients, cirrhosis developed within the first 2 years; all seven patients had bridging necrosis in the first liver biopsy, and five of these were infected with hepatitis delta virus. The remaining 13 (46%) patients did not develop cirrhosis during follow-up and showed either unchanged features of chronic active hepatitis (seven cases) or histologic improvement to chronic persistent hepatitis (five cases) or to normal liver (one case). In conclusion, the prognosis of anti-HBe-positive patients with chronic hepatitis B is poor, as 54% of the cases developed cirrhosis during a mean histologic follow-up period of 4.5 years, mainly in association with hepatitis delta virus infection or continuing hepatitis B virus replication.     

Characterization and biological properties of a hepatitis B virus isolated from a patient without hepatitis B virus serologic markers

We have developed a rapid method to characterize genomic diversity of low-level hepatitis B and related viral agents after their identification in serum by high-affinity HBsAg-antibody monoclonal antibody capture and subsequent polymerase chain reaction amplification. Serum from an individual with chronic liver disease and without hepatitis B virus serological markers but reactive by monoclonal antibody capture/polymerase chain reaction amplification was inoculated into a chimpanzee. After inoculation, an acute hepatitis B virus-like hepatitis developed in the chimpanzee. Analysis of serial liver biopsy samples showed the persistence of hepatitis B virus DNA for more than 17 mo after resolution of acute hepatitis and seroconversion. Applying the technique of restriction enzyme fragment analysis to serial chimpanzee liver biopsy samples and acute-phase sera, along with the serum inoculum, we established that all hepatitis B virus DNA sequences are derived from the same viral agent. We present evidence that the viral DNA persisted as a nonreplicating episomal form in the nucleus of hepatocytes. This study demonstrates that after clinical and serological recovery from an acute hepatitis, there may be persistence of low-level hepatitis B virus-related genome in the liver despite the presence of antibodies to HBsAg. Such persistence of viral genome may be a natural sequela of infection and may serve as a source of viral latency and possible reactivation. Finally, cloning and complete nucleic-acid sequencing of this virus have demonstrated multiple nucleotide and amino acid changes compared with all known hepatitis B virus subtypes. These changes may have contributed in part to a different antigenic composition or immunological reactivity of the host to this hepatitis B virus isolate.     

Acute hepatitis A infection in hepatitis B chimpanzee carriers

Two hepatitis B virus carrier chimpanzees which were superinfected with hepatitis A virus developed acute hepatitis followed by the production of antibodies to hepatitis A virus. The Southern blot technique employed to monitor liver hepatitis B virus DNA revealed that the amount of viral DNA in both animals was significantly reduced during the acute phase of hepatitis A infection. The levels of plasma hepatitis B DNA polymerase activity were also reduced in one chimpanzee. The high titers of HBsAg in the circulation remained unchanged throughout the study, and antibodies to the surface antigen and to e antigen were not detected. The morphological lesions in the liver were severe in one chimpanzee from whom one specimen showed both periportal focal necrosis and zonal parenchymal necrosis.     

Evidence for hepatitis B and other virus infection in HBsAg-negative chronic active hepatitis

Antibody to the hepatitis B core antigen (anti-HB_c) was detected in sera from 6 (19%) of 32 patients with HB_sAg-negative chronic active hepatitis. In three cases with the highest anti-HB_c titers, core antigen was detected in liver cell nuclei and in one case HB_sAg was also present in hepatocytes, suggesting continuing hepatitis B virus infection. During follow-up, anti-HB_c titers fell slowly in those with no viral antigens in liver tissue, and in two cases with virus in the liver at presentation, viral antigens were no longer demonstrable four and eight years later. In one case, clearance of virus was preceded by the appearance of HB_sAg in liver and serum, suggesting reactivation of viral replication. In three of the anti-HB_c-negative cases a nuclear antigen unrelated to the hepatitis B virus was detected by immunofluorescence, and it is possible that liver disease in these patients may be related to persistent non-A, non-B virus infection.     

Immunopathology of chronic viral hepatitis

This paper reviews the main necro-inflammatory liver lesions observed in chronic viral hepatitis B and their apparent immunological mechanisms. Focal necrosis seems to represent the mechanism for clearance of virus replicating hepatocytes by cytotoxic T lymphocytes under HLA class I restriction and with hepatitis B virus nucleocapsid antigens as target antigen. Piecemeal necrosis involves CD8+ lymphocytes, possibly with hepatocellular membrane autoantigens as target antigen. Confluent lytic necrosis (bridging hepatic necrosis) presumably results from humoral immune mechanisms. Focal necrosis, piecemeal necrosis and confluent lytic necrosis determine the extent of necro-inflammatory activity in liver biopsy specimens, classified as chronic persistent hepatitis (low activity) or chronic active hepatitis (high activity). The extent of necro-inflammatory activity fluctuates during the natural course of chronic viral hepatitis B: from low activity during the viral replicative phase, through high activity in the viral elimination phase, into low activity again in the ensuing viral integration phase. The role of HLA antigens, intercellular adhesion molecules, cytokines and accessory antigen presenting cells is emphasized.     

Characteristics of hepatitis B X antigen, antibodies to X antigen, and antibodies to the viral polymerase during hepatitis B virus infection

The characteristics of hepatitis B virus (HBV) X antigen (HBxAg) and antibodies against the X antigen (anti-HBx) and the viral polymerase (anti-pol) were determined in 85 HBV-infected patients. HBxAg was detected in sera positive for HBV e antigen (HBeAg) and HBV DNA in patients with acute and chronic hepatitis, while anti-HBx appeared when markers of viral replication became undetectable. HBxAg was common in the liver among patients with chronic hepatitis independent of HBV replication markers but was closely correlated with elevated alanine aminotransferase, implying that HBxAg in liver may be important in the pathogenesis of chronic infection. Anti-pol was detected in many samples positive for HBeAg and HBV DNA and less often in serum samples without markers of HBV replication, suggesting that this marker could reflect ongoing viral replication in the liver, even though such markers were absent from sera.     

Latent hepatitis B virus infection with full-length viral genome in a patient serologically immune to hepatitis B virus infection

The presence, state, physical structure and cellular localization of hepatitis B virus (HBV) DNA were investigated in a patient with hepatitis B surface antigen (HBsAg)-negative chronic liver disease. HBV serology was positive for antibodies to hepatitis B core antigen (anti-HBc), to hepatitis B e antigen (anti-HBe) and to HBsAg (anti-HBs); no HBV DNA was detectable in serum. Southern blot analyses of DNA extracted from the liver demonstrated free monomeric HBV DNA as two distinct species: a predominant species of fully double-stranded relaxed circular molecules and a minor species of linear molecules of 3.2 kilobase pairs (kbp) length. Restriction enzyme analyses identified the HBV genome as HBsAg subtype adw2. Cell fractionation studies further revealed that the free viral DNA species were localized exclusively in liver cell nuclei. These findings in a patient serologically immune to HBV infection demonstrate that in hepatocytes HBV can establish a latent infection, characterized by the extrachromosomal presence of a full-length viral genome without production of infectious virus or synthesis of viral antigens.     

Immunoglobulin A antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus

Antibody to hepatitis B core antigen of immunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio greater than 2.1, was detected in all of 39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected in only 2 (4%) of 46 asymptomatic carriers of the virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean +/- SE titer of antibody in chronic persistent hepatitis (3.8 +/- 0.9) was significantly lower than those in chronic active hepatitis (13.8 +/- 3.2) and cirrhosis with or without carcinoma (25.6 +/- 6.1) (p less than 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection.     

Reactivation of chronic type B hepatitis presenting as acute viral hepatitis

Three asymptomatic chronic carriers of hepatitis B surface antigen, who had normal serum aminotransferase levels and no detectable hepatitis B e antigen in serum, developed icteric, symptomatic acute hepatitis. Serologic evidence of acute infection with hepatitis A virus, delta hepatitis virus, cytomegalovirus, or Epstein-Barr virus was absent. However, hepatitis B virus DNA and DNA polymerase activity, which were not detectable before the exacerbation, appeared in the serum of all three patients during the acute illness, confirming the diagnosis of spontaneous reactivation of chronic type B hepatitis. Thus, acute exacerbations of chronic type B hepatitis may present as an acute hepatitis superimposed on the chronic carrier state.     

Human genes involved in hepatitis B virus infection

Persistent hepatitis B virus(HBV)infection is a significant public health problem because it is a major cause of chronic liver disease,cirrhosis,and hepatocellular carcinoma(HCC).Roughly one-third of the world population has been infected with HBV and there are about350 million(5%-6%)persistent carriers.HBV causes80%of all liver cancer cases and is the second most important carcinogen,after smoking tobacco.There is an approximate 90%risk of becoming a persistent carrier following perinatal infection in infants born to e antigen positive carrier mothers and a 30%risk in pre-school children.Only 5%-10%of adults become persistent carriers following infection.Of individuals persistently infected with HBV,10%-30%will develop liver cirrhosis and HCC.These highly variable outcomes in both clearance rates and disease outcomes in persistently infected individuals cannot be fully explained by differences in immunological,viral or environmental factors.Thus,differences in host genetic factors may affect the natural history of hepatitis B.     

Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection

The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA (P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease (P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer (P = 0.0017), whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.     

Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis

Serum samples from 130 persons who were seropositive for hepatitis B surface antigen and who had various forms of accompanying liver disease were tested for immunoglobulin M (IgM) antibody to hepatitis B core antigen. In 99% of patients with hepatitis B antigen-positive chronic type B hepatitis, IgM antibody to hepatitis B core antigen was present. This antibody was not present in "healthy" hepatitis B surface antigen carriers and was detectable in only 30% of patients with delta hepatitis. Testing of serial sera from 38 patients with chronic type B hepatitis revealed that IgM antibody to hepatitis B core antigen persisted in patients who had evidence of persistent hepatitis B virus replication but ultimately disappeared in those patients who exhibited a sustained loss of serum markers of viral replication (hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase activity). These findings suggest that the presence of IgM antibody to hepatitis B core antigen in chronic hepatitis B surface antigen carriers indicates an active immune response to persistent viral replication.     

Hepatitis B virus deoxyribonucleic acid in liver of chronic carriers. Correlation with serum markers and changes associated with loss of hepatitis B e antigen after antiviral therapy

Patients with chronic hepatitis B receiving antiviral or immunomodulatory therapy were prospectively studied to determine the relationship between the state of hepatitis B virus deoxyribonucleic acid in liver and the levels of serum markers of hepatitis B virus infection. Hepatitis B virus deoxyribonucleic acid was quantitated and the molecular forms determined using molecular hybridization in two separate liver specimens taken at least 1 yr apart. All 30 patients initially had hepatitis B surface antigen and e antigen in serum and all had hepatitis B virus deoxyribonucleic acid, including replicative intermediate forms, present in liver. The amount of viral deoxyribonucleic acid in liver correlated significantly with the e antigen titer in serum. At the time of the second liver biopsy, e antigen was no longer detectable in the serum of 12 patients although all except 1 patient still had detectable hepatitis B surface antigen. In this group, the amount of hepatitis B virus deoxyribonucleic acid in liver had decreased significantly and replicative viral intermediates had disappeared. In contrast, among patients who remained e antigen-positive, the amount of viral deoxyribonucleic acid did not change appreciably and replicative intermediate forms were still detectable. These findings imply that in chronic hepatitis B, loss of e antigen is usually associated with resolution of hepatitis B virus replication.     

Correlation of e antigen, DNA polymerase activity, and Dane particles in chronic benign and chronic active type B hepatitis infections.

The e determinant of hepatitis B surface antigen (HBS Ag) was found in 23 of 42 patients with chronic hepatitis B virus (HBV) infection. Presence of e antigen was associated with increases in DNA polymerase activity and in the number of circulating Dane particles. In the group with detectable e antigen, the average DNA polymerase activity was 367+/-78 counts per minute (cpm; mean+/-standard error [SE]), and the average number of Dane particles counted in electron micrographs was 4.4% of the total HBS Ag. In contrast, e antigen-negative patients had an average DNA polymerase activity of 40+/-6.9 cpm (P less than 0.1) and an average Dane particle count equal to 0.6% of the HBS Ag. The e antigen was detected in 68% of patients who were HBS Ag carriers or had persistent viral hepatitis and 40% of those with chronic active type B hepatitis. Thus, the presence of e antigen correlated with both the chronicity and presence of infectious HBV. However, it did not correlate with the type or severity of liver disease after HBV infection, since e antigen was present in both chronic benign and chronic aggressive hepatitis B infections.     

Interferon alfa therapy in patients with chronic hepatitis B virus infection. Effects on hepatitis B virus DNA in the liver

Pretrial and posttrial liver biopsy samples from 124 adult patients who participated in two randomized, controlled trials of interferon alfa therapy for chronic hepatitis B virus (HBV) infection were analyzed to determine the effects of interferon on the replication of HBV in the liver. Replicative forms of HBV DNA were detected in the pretrial biopsy samples from all and posttrial biopsy samples from 74% treated patients and 86% controls. Replicative forms of HBV DNA were detected in the posttrial biopsy samples from all patients who remained positive for hepatitis B e antigen and HBV DNA in the serum, in 77% treated patients and 80% controls who cleared HBV DNA in the serum but who remained positive for hepatitis B e antigen, but in only 19% treated patients and 40% controls who cleared HBV DNA as well as hepatitis B e antigen in the serum. Serum alanine aminotransferase levels were significantly lower in patients whose posttrial biopsies did not contain replicative forms of HBV DNA. In summary, we demonstrated that in most patients with chronic HBV infection treated with interferon alfa, serological response was associated with the disappearance of replicative forms of HBV DNA in the liver.     

Natural history of chronic hepatitis B virus infection: New light on an old story

Chronic hepatitis B virus (HBV) infection is one of the most common persistent virus infection in man. It causes significant morbidity and mortality, and therefore is important. Extensive studies on clinicopathologic studies and long-term follow up on hepatitis B surface antigen (HBsAg) carriers have largely disclosed the natural history of chronic HBV infection. The infection easily becomes chronic when contracted in early infancy. As high as 90% of babies born to HBV carrier mothers will also become HBsAg carriers. Once chronic infection is established, it is refractory, and HBsAg carriage usually persists for life. However, the chronic infection is not monotonous, it actually evolves from an HBV replicative phase to a non-replicative phase. The host responds differently and with more complexity in different phases. The virus-host interactions, divided into three phases, virus tolerance, virus clearance and residual HBV integrated phases, result in a heterogeneous variety of hepatic lesions. The first two phases occur when HBV is actively replicating, and the last corresponds to the non-replicative phase. The high HBV level (and hence HBV gene products) renders the host's immune system tolerant to the virus, and the infected host does not exert an effort to get rid of the virus. At this stage, the liver is nearly normal, and the host is asymptomatic. However, later in the replicative phase, the HBV replication begins to wane, and the immune tolerance is no longer maintained. Hepatitis B core antigen/hepatitis B e antigen (HBcAg/HBeAg)-specific cellular immune responses result in lysis of the infected liver cells; the liver then begins to have active disease as revealed by the presence of lobular hepatitis. The asymptomatic carrier may then start to have symptoms of hepatitis. After a variable period, usually in years, the host eventually gets rid of active viral replication and only residual incomplete HBV genome integrated to host chromosomes is found. The carrier is now HBeAg negative/anti-HBe positive, serum HBV DNA decreases to very low levels, and the disease becomes qulescent at this stage. The outcome of the host is determined by the hepatic lesions caused by HBV-host interactions mentioned above, with cirrhosis and hepatocellular carcinoma (HCC) as the major sequelae of chronic HBV infection. Although HCC is usually preceded by HBV-induced cirrhosis, this is not always the case. Cirrhosis and HCC may develop independently, with cirrhosis as the most important precipitating factor or cofactor of HCC. A significant proportion of HBsAg carriers, particularly the males, will eventually die of these sequelae.