Hepatitis B virus with X gene mutation is associated with the majority of serologically "silent" non-b, non-c chronic hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically "silent" non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically "silent" NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.     

Prevalence of mutations in core promoter/precore region in Japanese patients with chronic hepatitis B virus infection

We examined the frequency and significance of mutations in the core promoter and precore region in 103 Japanese patients with chronic hepatitis B virus (HBV) infection. HBV DNAs from the patients' sera were amplified by polymerase chain reaction and were directly sequenced. A double mutation (T1762 A1764) in the core promoter was frequently observed in the patients regardless of HBeAg status except for asymptomatic carriers with HBeAg. Furthermore, a mutation at nucleotide 1753 from T to C or G was frequently found in anti-HBe positive patients and was often accompanied by the double mutation. The A1896 mutation was found in only about one fourth of the patients with anti-HBe. These data suggest that the patients with chronic liver diseases frequently had a double mutation regardless of HBeAg status and a mutation at nucleotide 1753 might be associated with HBeAg-negative chronic hepatitis B virus infection.     

Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan

It was recently reported that mutations in the precore and core promoter region of hepatitis B virus (HBV) are associated with fulminant hepatitis. The aim of this study was to investigate the association of mutations in the precore and core promoter region of HBV with fulminant and severe acute hepatitis. We studied Japanese patients with acute HBV infection, including seven patients with fulminant hepatitis, 12 with severe acute hepatitis and 41 with acute self-limited hepatitis. The presence of HBV mutants was examined by using a point mutation assay to detect a G to A transition at position 1896 in the precore region and an A to T transition at position 1762 and a G to A transition at position 1764 in the core promoter region. Significant differences in the proportion of mutations in the precore or core promoter region were present between patients with fulminant hepatitis and self-limited acute hepatitis (7/7 (100%) vs 4/41 (9.8%), P<0.01) and between severe acute hepatitis and self-limited acute hepatitis (6/12 (50.0%) vs 4/41 (9.8%), P<0.01). The frequency of mutation increased proportionately with the severity of disease in patients with acute HBV infection. Fulminant hepatitis B in Japan is closely associated with mutations in the core promoter and precore gene of HBV. Point mutation assays for HBV precore and core promoter analysis may be useful to predict the outcome of liver disease in patients with acute HBV infection.     

Functional analysis of HBV genomes from patients with fulminant hepatitis

Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild-type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild-type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild-type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.     

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-alpha therapy (n = 37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.     

Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in transgenic mice

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.     

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIM: During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon. METHODS: Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced. RESULTS: In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found. CONCLUSION: HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.     

Spontaneous seroconversion in chronic hepatitis B: role of mutations in the precore/core gene

The mechanism underlying spontaneous clearance of hepatitis B e antigen (HBeAg) and the appearance of antibodies (anti-HBe seroconversion) in chronic hepatitis B is not known. Previous studies have demonstrated mutations within the precore/core gene before, during, and after seroconversion, suggesting that the emergence of mutations in the core gene may abrogate tolerance and that this event may act as a general principle for the initiation of the clearance of HBeAg. To investigate this hypothesis, we studied three patients with adult-acquired chronic hepatitis B virus (HBV) infection before spontaneous seroconversion by sequential sequencing and single-stranded conformation polymorphism (SSCP) analysis of the entire precore/core genome. In one patient, a new viral strain appeared six months before seroconversion, but no mutations or new viral strains could be detected in the other two patients. SSCP analysis confirmed the sequencing results and revealed no evidence for the emergence of new viral subpopulations before seroconversion. These results suggest that the appearance of nucleotide changes within the precore/core region of the dominant viral strain is not a prerequisite for the induction of seroconversion in patients with chronic hepatitis B virus infection acquired during adulthood.     

Relativistic density-dependent Hartree-Fock approach for finite nuclei

Two precore predominant mutations of human hepatitis B virus (HBV) at either nucleotide (nt) 1896 or nt 1899 often occur in combination. At nt 1896, a G to A mutation creates a TAG stop codon at codon 28 of precore protein. At nt 1899, a G to A mutation changes glycine at codon 29 to aspartic acid. To assess the effect of each individual mutation as well as any interaction between these two mutations, HBV derivatives bearing one or both precore predominant mutations have been constructed. HBV e-Ag-negative mutants bearing a TAG stop codon mutation at codon 28 uniformly replicate at least 20-fold better than mutants bearing a TGA stop codon at the same amino acid position, irrespective of the sequence context at nt 1899. A single mutation at nt 1899, changing the wild-type G to a pyrimidine (T or C) is deleterious to viral RNA encapsidation and DNA replication. Our results explain in part why only a purine (G or A) at nt 1899, never a pyrimidine, is observed in natural HBV genomes. The effects caused by these two closely linked mutations on viral replication are not independent of each other. The stringent selection for a highly efficient RNA encapsidation element may play a crucial role in the natural occurrence of these two closely linked precore mutations. The putative 27-amino-acid peptide resulting from the truncation of precore by the nt 1896 mutation has no apparent effect on viral replication. The preferential occurrence of the G to A mutation at nt 1896 and 1899, instead of at other nonpredominant positions, is likely to be a combined consequence of both selection and higher intrinsic mutation frequency at these positions.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

The intrahepatic expression of the hepatitis B virus in chronic delta hepatitis

In order to evaluate the interference of hepatitis delta virus (HDV) in hepatitis B viral particle (HBsAg, HBcAg) expression in the liver of chronic HDV patients, 39 and 81 liver biopsies of HBsAg carriers seropositive for anti-HDV and anti-HDV negative controls, respectively, were studied. HBcAg was positive in 16.7% of the HBeAg-positive patients with HDAg in the liver and in 91,4% of controls. In contrast, in HBeAg- and anti-HDV negative patients the intrahepatic expression of HBcAg was detected in 32.6%. In anti-HDV negative patients the HBcAg liver expression correlated significantly with the HBeAg in serum (p < 0.00001). The distribution of HBcAg was exclusively cytoplasmatic in 30% of HDV-infected patients but mixed nuclear and cytoplasmic in 38.3% of the controls. The nuclear expression of HBcAg was decreased in chronic HDV infection. HBsAg was positive in 70.3% of patients who were anti-HDV positive and in 82.3% of controls. The membranous expression of HBsAg was detected less frequently in HDV-infected patients (p < 0.05) than in controls, while associated with HBeAg in serum of HBV carriers without HDV superinfection (p < 0.00001). The prevalence and the HBsAg cytoplasmic expression was not different for the chronic HDV infection or controls. Our results show: 1) decreased intrahepatic expression of HBcAg and membranous HBsAg in HBV carriers superinfected with HDV, suggesting decreased HBV replication in the liver of these patients. 2) the changing of HBcAg and HBsAg expression in the liver of HDV-infected patients, suggest not so much a decrease but rather a modulation in HBV replication.     

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.     

Interleukin-12 induction of Th1 cytokines is important for viral clearance in chronic hepatitis B

Interleukin-12, a cytokine with an important role against intracellular pathogens, promotes Th1 cell development, cellmediated cytotoxicity, and interferon-gamma production. We investigated the immunoregulatory role of IL-12 in 72 chronic hepatitis B virus (HBV) carriers, 33 of whom were monitored longitudinally during interferon-alpha treatment. Serum levels of IL-12 heterodimer, IL-12 p40 subunit, IL-4, and Th1 cytokines were determined by specific ELISAs, and hepatitis B core antigen-specific T cell response by a proliferation assay. Chronic HBV carriers had higher serum levels of IL-12 and IL-12 p40 in comparison with controls (P < 0.01), suggesting that IL-12 production is not impaired. The longitudinal analysis revealed a further substantial increase (> 2.5x baseline level) of bioactive IL-12 and Th1 cytokines in patients who cleared HBV and seroconverted to anti- hepatitis B e, unlike the 23 nonresponders with persistent HBV replication (P < 0.01). The IL-12 peak followed the peak of hepatocytolysis by 9.8+/-2.8 wk and occurred either before or simultaneously with hepatitis B e seroconversion. Hepatitis B core antigen-specific T cell proliferation closely correlated with hepatocytolysis and increased significantly in all patients (8 responders and 15 nonresponders) who developed hepatitis flare, irrespective of the virological outcome. These results provide in vivo evidence that IL-12 may have an important role for viral clearance in chronic HBV infection.     

Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.