Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

Evolution of viral load and changes of polymerase and precore/core promoter sequences in lamivudine-resistant hepatitis B virus during adefovir therapy

Adefovir dipivoxil (ADV) has demonstrated clinical activity against both wild-type and lamivudine-resistant hepatitis B virus (HBV). We analyzed the evolution of viral load and the changes of polymerase and precore/core promoter sequences in lamivudine-resistant virus during ADV therapy. The authors studied 14 patients who had breakthrough hepatitis after lamivudine therapy. Serial sera were obtained prior to adefovir administration and at 3, 6 and 12 months after ADV therapy. Nucleotide sequences of polymerase and the precore/core promoter from the hepatitis B virus were analyzed. The median serum HBV DNA decrease with adefovir treatment was 4.35 log(10) copies/mL at 12 months. Tyrosine-methionine-aspartate-aspartate (YMDD) mutants were found in 12 patients among the 14 patients with lamivudine resistance. The YMDD mutant viruses reversed to the wild-type in 6 patients out of the 12 patients after 3-6 months of ADV after discontinuing lamivudine therapy. In the analysis of the nucleotide sequences of the precore/core promoter gene, core promoter mutants in 12 patients were replaced by wild-type virus in three patients (25%), while precore mutants in four patients were replaced by the wild-type in three patients (75%). The results demonstrate the patterns of polymerase and precore/core promoter mutations in lamivudine-resistant hepatitis B viruses and the reversion from the mutant to the wild-type in some patients. In addition, despite several mutations in the polymerase during ADV therapy, ADV effectively suppressed HBV replication without the emergence of resistant viral mutants.     

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: Does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)-positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA-positive samples were subjected to polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) to screen mutations in the Precore, core, and the X-gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA-positive. Of the 40 HBV DNA-positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg-positive patients, as compared to HBeAg-negative patients, over 1 year. At follow-up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re-designated as DNA-negative on follow-up and had wild-type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow-up, there was evidence of spontaneous reduction in the mean viral load over the 1-year study period.     

Restoration of replication phenotype of lamivudine-resistant hepatitis B virus mutants by compensatory changes in the "fingers" subdomain of the viral polymerase selected as a consequence of mutations in the overlapping S gene

The introduction of lamivudine (LMV) for the treatment of chronic hepatitis B infection has been an important advance in the management of this disease. However, the long-term efficacy of LMV may become limited by the emergence of antiviral-resistant hepatitis B virus (HBV) mutants. The two most common LMV-resistant mutants produce changes in the viral polymerase protein (rt) of rtM204I and rtL180M/M204V (previously rtM550I and rtL526M/M550V). A number of studies have demonstrated that these HBV mutants appear to be replication impaired, both in vitro and in vivo. The detection and selection of compensatory mutations in the polymerase protein that restore the replication phenotype of these HBV mutants have been poorly described to date. The effects of mutations in the fingers subdomain of the viral polymerase protein arising as a consequence of vaccine and hepatitis B immune globulin (HBIg) selected changes in the overlapping envelope gene (S), and a determinant of the hepatitis Bs antigen (HBsAg) were analyzed in vitro. The LMV-resistant HBV mutants rtM204I and rtL180M/M204V produced substantially weaker HBV DNA replicative intermediate signals by Southern blot analysis and less total intracellular HBV DNA by real-time PCR compared to wild-type virus. The viral polymerase protein of these mutants produced little detectable radiolabeled HBV DNA in an endogenous polymerase assay. In contrast, the HBV a determinant HBIg/vaccine escape mutants sP120T, sT123N, sG145R, and sD144E/G145R (that produce rtT128N, Q130P, rtW153Q, and rtG153E respectively) yielded as much virus as wild-type HBV while the sM133L (rtY141S) mutant was replication impaired. Two of these mutants, rtT128N and rtW153Q, when introduced into a replication-competent HBV vector containing the rtL180M/M204V polymerase mutation restored the replication phenotype of this LMV-resistant mutant. These viruses produced levels of intracellular HBV DNA as determined by Southern blot and real-time PCR that were comparable to those of wild-type HBV, indicating that the changes in the fingers subdomain were able to compensate for the reduced replication of the LMV-resistant mutations. Since these viruses carry mutations in the a determinant of HBsAg that may potentially decrease the ability of anti-HBs antibody to neutralize these viruses, these HBV mutants also have the potential to behave as vaccine escape mutants.     

Hepatitis B virus reactivation in patients undergoing cytotoxic chemotherapy for solid tumours: precore/core mutations may play an important role

Reactivation of the hepatitis B virus (HBV) is a rare, but well described complication of cytotoxic chemotherapy that may result in hepatic failure. Patients who are chronic carriers of the HBV and who have a G to A mutation at nucleotide 1896 in the precore region may develop more severe liver disease, possibly because of rapid selection and enhanced replication ability of the mutant strain. Such mutant viruses have been implicated occasionally in chemotherapy induced reactivation of hepatitis B virus. In this report, 5 patients with solid tumours were identified to have developed severe hepatitis B virus related liver disease during treatment with cytotoxic agents (with dexamethasone as anti-emetic). All had clinical and serological evidence of reactivation of the HBV. Three patients developed icteric hepatitis; 2 fully recovered, and 1 had died from progressive metastatic disease while recovering from the reactivation. The other two died from progressive liver failure. Direct sequencing of the polymerase chain reaction (PCR) products of the precore (preC) and precore promoter region of the HBV-DNA was carried out on the patients' serum samples taken during the episode of reactivation. In each case, similar mutations (G to A) in nucleotide 1896 of the preC region were found, together with additional mutations in the preC promoter. The present findings suggest that reactivation involving a mutant hepatitis B virus may lead to liver failure, which is possibly more severe than that caused by wild type HBV, and can be triggered by cytotoxic chemotherapy, or the administration of corticosteroids. In Eastern Asia the HBV carriage rate in adults is high. HBV reactivation and severe liver disease during cytotoxic treatment may become a serious and common problem in this region as cytotoxic chemotherapy is more widely used. Patients should be screened routinely for HBsAg in endemic areas of chronic hepatitis B virus infection prior to receiving cytotoxic treatment. The possibility of HBV reactivation should be considered in patients developing liver dysfunction. Patients who are HBeAg negative/Anti-HBe positive, and are suspected to be having an HBV reactivation, should have HBV-DNA levels measured for confirmation as they may carry a mutant HBV.     

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.     

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.     

Analysis of the Hepatitis B virus precore and ORF-X sequences in patients with antibody to hepatitis B e antigen with and without normal ALT levels

Serum samples from 20 anti-hepatitis B e antigen-positive patients with and without normal alanine aminotransferase (ALT) levels who had serum hepatitis B virus (HBV) DNA detectable only by polymerase chain reation (PCR) were examined. Viral DNA was amplified by PCR, using primers that encompassed precore and ORF-X regions and sequenced directly, to investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity of disease and in the levels of viral replication. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT. The amount of HBV-DNA correlated with the ALT levels (P < 0.05). Seventy-two percent of patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBV-DNA concentration (P < 0.05). Thirty percent of patients had mutations in ORF-X. Patients with ORF-X mutations had lower levels of HBV-DNA than those who had wild-type virus. The presence of mutations in precore and X regions may be related to a low HBV-DNA concentration and reduced biochemical activity in patients with anti-HBe. J. Med. Virol. 56:294–299, 1998 . © 1998 Wiley-Liss, Inc.     

Evolution of wild-type and precore mutant HBV infection after liver transplantation

Recurrence of hepatitis B virus (HBV) infection after liver transplantation is associated with varying degree of graft damage. The aim of the study was to investigate longitudinally the changes of wild-type and precore A₁₈₉₆HBV mutant viral populations after reinfection and their impact on liver graft damage. The wild-type HBV and A₁₈₉₆HBV strains were quantitated before and serially after orthotopic liver transplantation (OLT) in 14 hepatitis B surface antigen (HBsAg)-positive liver graft recipients (4 hepatitis B e antigen [HBeAg]+; 10 anti-HBe+). Before OLT, the wild-type precore HBV was present in all 4 HBeAg-positive patients and in 2/10 anti-HBe-positive patients; a mixed virus population was present in 6 patients; and A₁₈₉₆HBV mutant alone in 2 patients. After OLT, A₁₈₉₆HBV mutant appeared and gradually accumulated in 5/6 patients who had the wild-type HBV before OLT and 1 of these patients seroconverted from HBeAg to anti-HBe 52 months after transplantation. A mixed HBV population was present continuously in 6 patients before and after OLT. Of the 2 patients with A₁₈₉₆HBV only pre-OLT, the wild type appeared in one patient and the other patient retained persistently the A₁₈₉₆HBV mutant. There was no relationship between liver graft histology and the type of viral population at reinfection or at the end of follow up. Changes in the HBV population occur during follow up of recurrent hepatitis B in liver transplant recipients with frequent accumulation of precore A₁₈₉₆HBV mutants, but the type of viral population does not determine the severity of hepatitis B in the graft. J. Med. Virol. 59:5–13, 1999. © 1999 Wiley-Liss, Inc.