A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10 3 U/ml, all healthy control (HBsAg/HBV-DNA negative; n ؍ 108) and anti-HCV antibody-positive (n ؍ 59) sera were identified as negative. The assay showed a detection limit of 4 ؋ 10 2 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification ( Many hepatitis B virus (HBV) markers are used for diagnosing and monitoring hepatitis B patients. HBV-DNA tests, such as the branched-chain DNA (b-DNA) signal amplification assay (7, 31), and transcription-mediated amplification (TMA)-based (11) or PCR-based (12,14,20) assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skill and high costs. These gene amplification assays also present some limitations (22,23,35). The b-DNA assay provides quantitative results but requires a long incubation time and lacks adequate sensitivity. Amplification assays have adequate sensitivity but are less quantitative.Immunoassays are generally easy and inexpensive. There have been a few reports of serum HBcAg assays with specimen pretreatment (4, 32). The concentration of HBcAg in these assays correlated with levels of HBV-associated DNA polymerase (4). Thus, HBcAg could be a marker for virus load. However, the use of these assays is limited because of relatively low sensitivity and complex procedures.Serum HBeAg concentration reflects virus replication and hepatitis activity and is closely correlated with virus load in anti-HBe antibody-negative patients (8). Seroconversion of HBeAg to anti-HBe antibody reveals the inactive phase of infection (17,25). However, after seroconversion, many patients may exhibit reactivation and high viral load (3,10,18). In these cases, HBeAg is usually negative due to masking by anti-HBe antibody (24), although the HBeAg/anti-HBe immune complex can be indirectly detected according to the levels of alanine aminotransferase (ALT) and HBV-DNA (6). Therefore, HBcAg and HBeAg could be expected to be efficient markers of virus load if antibodies were inactivated and the antigens released.In the present study, for the purpose of developing a simple, sensitive, and inexpensive assay for determining HBV virus load, we targete...
A retrospective survey of Japanese patients histologically diagnosed with chronic hepatitis B was conducted to determine the effectiveness of lamivudine in preventing hepatocellular carcinoma (HCC). Of the 2,795 patients who satisfied criteria for analysis after treatment from any of 30 medical institutions, 657 had received lamivudine and the remaining 2,138 had not. A Cox regression model with liver biopsy as the starting point revealed seven factors related to HCC: lamivudine therapy, gender, family clustering of hepatitis B, age at liver biopsy, hepatic fibrosis stage, serum albumin level, and platelet count. In a matched case-controlled study, 377 patients in a lamivudine-treated group and 377 matched patients in a non-treated group were selected based on their propensity scores. The mean follow-up period was 2.7 years in the lamivudine group and 5.3 years in the control group. In the lamivudine group, HCC occurred in 4 patients (1.1%) with an annual incidence rate of 0.4%/patient/year, whereas in the control group HCC occurred in 50 patients (13.3%) for a rate of 2.5%/patient/year. A comparison of the cumulative HCC incidence between the two groups by the Kaplan-Meier method showed a significantly lower incidence of HCC in the lamivudine group (p<0.001). These findings suggest that lamivudine effectively reduces the incidence of HCC in patients with chronic hepatitis B.
DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera.The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/ core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles. Hepatitis B virus (HBV)1 infects more than 300 million people and is a major cause of liver diseases. The HBV belongs to the Hepadnavirus family and is a small (42 nm) enveloped DNA virus, which possesses a 27-nm icosahedral nucleocapsid composed of core protein and a 3.2-kb partially doublestranded, circular genome (1). Although the term "Dane particles" refers to the 42-nm HBV particles (2) and is often used in reference to the complete HBV particles, electron microscopic studies have suggested that the DNA-negative "empty" Dane particles are predominant in sera (3-6). The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail.The HBV genome encodes two core-related open reading frames, precore and core genes (Fig. 1). These are expressed because of two in-frame ATG initiation codons located at the 5Ј end of the genes. The first ATG encodes a 25-kDa protein (p25) containing the 29-amino acid (aa) precore sequence fused to the N terminus of the HBV core antigen (HBcAg). The p25 is directed toward the secretory pathway by a 19-aa signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER), producing a 22-kDa protein. Subsequent proteolytic cleavages within the arginine-rich Cterminal region (34 aa) generate a 17-kDa protein that is secreted as hepatitis B e antigen (HBeAg) (7-10). A heterogeneous population of these precore derivatives has been observed in the sera of patients and is serologically defined as HBeAg (9,11,12). Conversely, the second ATG specifies the 21.5-kDa HBcAg, which assembles into dimers that form the virus capsid (7,9,(13)(14)(15). HBcAg is a 183-residue protein wi...
We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core-related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV-negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription-mediated amplification assay (TMA) and in-house real-time detection polymerase chain reaction (RTD-PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P < 0.001) over a range which varied 100 000-fold. The accuracy in the measurement of the patients' HBV load obtained using the HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti-viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patient's HBV load. When monitoring the anti-viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.
correlated both with levels of HBV DNA and HBVcrAg at the beginning and 2 months after the start of lamivudine therapy. Conclusions. HBV RNA is detectable in serum in a form incorporated in virus particles, and its serum level is possible to be a new viral marker with different significance than HBV DNA in lamivudine therapy.
A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 g/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n ؍ 160) and anti-hepatitis C virus-positive (n ؍ 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r ؍ 0.946, n ؍ 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.Diagnosis of chronic hepatitis B virus (HBV) infection has long been based on HBV serology and measurement of liver enzymes. With the development of therapies for chronic HBV infection including interferon and lamivudine (9, 16), quantitative detection of HBV DNA has been used increasingly as the most important marker for monitoring HBV replication activity and disease progression as well as for assessing responses to antiviral treatment of patients with chronic hepatitis B (7,8). Several assays for the quantitative measurement of HBV DNA have been developed, such as the branched-chain DNA signal amplification assay (5, 7, 28) and transcriptionmediated amplification (TMA)-based (10) or PCR-based (6, 11, 13, 17) nucleic acid amplification assays. However, these methods tend to generate highly divergent results (20,21,22,31) and require cumbersome procedures and expensive equipment, in turn requiring considerable skill and high costs.On the other hand, immunoassays are generally easy and inexpensive. The nucleocapsid of HBV is composed of either 90 or 120 dimers of HBV core antigen (HBcAg) (3), released into circulation after envelopment. Hence, the quantity of HBcAg in serum would demonstrate virus load as well as HBV DNA. Serum HBcAg assays with specimen pretreatment have been reported previously (4, 29), and the concentration of HBcAg in these assays correlated with levels of HBV-associated DNA polymerase (4). Thus, HBcAg could be a marker for virus load.However, the use of these assays was limited because of relatively low sensitivity and complexity in the procedures.We have developed an enzyme immunoassay (EIA) for hepatitis B virus c...
Factors influencing and predictive of seroconversion from hepatitis B e antigen (HBeAg) to antibody (anti-HBe) were sought in a case-control study of 61 patients with chronic hepatitis B who had been observed from 5 years before to 1 year after seroconversion, and 32 patients who did not seroconvert during the entire 6-year period. Almost all of the patients (96%) were infected with HBV genotype C. HBV DNA levels began to decrease 3 years before seroconversion in the seroconverters, while they remained high in the non-converters. The frequency of precore mutation and the loss of HBeAg (A1896) started to increase 1 year before in the converters, and became significantly higher at seroconversion (23 vs. 3%, P = 0.030) than that in the non-converters. Double mutation in the core promoter (T1762/A1764) was more common in the seroconverters than in the non-converters 5 years before seroconversion (48 vs. 28%), and became significantly more frequent at seroconversion (65 vs. 41%, P = 0.046). Seroconversion occurred in 75% of the patients with at least HBV DNA levels <5.5 logarithmic equivalents/mL; precore mutation in 20% or more of HBV DNA; or core promoter mutation. Seroconversion occurred in 50% of those patients within 1 year, 88% within 2 years, and 93% within 5 years. These results indicate that a decrease in HBV DNA levels and mutations in the precore region and the core promoter were associated significantly and complementarily with seroconversion, and each of them or a combination thereof was predictive of seroconversion years ahead.
De novo infection of hepatitis B virus (HBV) occurs after liver transplantation from donors with HBV markers that suggest past infection. In the present study, the complete nucleotide sequences of HBV derived from a donor and recipients were determined to determine the clinical and virological characteristics. A total of 57 donor-recipient pairs, which underwent living-related orthotopic liver transplantation, were enrolled in the present study; all were negative for HBsAg before transplantation. HBV DNA was tested in serum, liver tissue, and peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR). The nucleotide sequence of HBV was determined based on PCR products and the phylogenetic analysis. De novo infection of HBV was found in 3 of the 57 recipients. Anti-HBc was positive in all donors of 3 recipients with the de novo infection but was positive only in 4 donors of the remaining 54 recipients (P=0.001). HBV DNA was detected in the liver but not in the serum or PBMCs in donor 3 whose recipient developed de novo HBV infection. The nucleotide sequence covering entire genome of HBV (3,215 bases) derived from the liver of donor 3 had a homology of 99.8-100% with that derived from the serum of corresponding recipient 3. The strain of recipient 3 showed the closest association with that of the donor 3 by phylogenetic analysis. Complete sequences from two recipients with de novo HBV infection including recipient 3 conserved the basic organisation of HBV genome. Analysis of the entire nucleotide sequence of HBV genome proved that HBV existed in the liver of the donor with anti-HBc, and it caused de novo infection in the corresponding recipient.
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