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 precore and core promoter DNA sequence in liver tissues from patients with hepatocellular carcinoma.

To investigate the role of mutant hepatitis B virus (HBV) in the development of hepatocellular carcinoma (HCC), 20 patients with HCC were studied for precore and core promoter mutations in tumorous and nontumorous tissues. The precore and core promoter region was amplified and analyzed by direct sequencing. Among the 20 tumorous and nontumorous tissues, precore mutant HBV was found in 12 (60%) and 18 (90%), respectively. Of the 12 tumorous tissues with precore mutant, nine tissues had a single mutation (1896) and one tissue had another single mutation (1899). The remaining two tissues had a double mutation (1896 and 1899). A single mutation (1896) and a single mutation (1899) were found in 11 and two of the 18 nontumorous tissues with precore mutant, respectively. Among 20 tumorous and nontumorous tissues, HBV with a C to T mutation at nucleotide (nt) 1846 was detected in six and eight, respectively, and was associated with the virus carrying a mutation (1896 or 1899) except in two tumorous tissues. Mutations at nt 1762 and 1764 in core promoter were observed in 16 (80%) tumorous tissues and 18 (90%) nontumorous tissues. Mutations in the precore and core promoter region were found frequently in nontumorous tissue and in tumorous tissue (18/20 and 12/20 in precore region, 18/20 and 16/20 in core promoter respectively). The high prevalence of precore and core promoter mutations in liver tissue from patients with HCC suggests that these mutations may contribute to the development of HCC.     

In vitro replication phenotype of a novel (-1G) hepatitis B virus variant associated with HIV co-infection

The -1G mutant HBV is more prevalent in individuals co-infected with HIV/HBV than in individuals infected with HBV alone and in some cases is the dominant virus in circulation. This mutant is created by the deletion of a dGMP (-1G) from the guanine rich homopolymer sequence located at nts 2,085-2,090 (numbering from EcoRI site as position 1) in the HBV core gene. This deletion causes a frameshift generating a premature stop codon at (64) Asn in the HBV core gene (codon 93 in the precore gene), that truncates the precore protein, precursor of the secreted hepatitis B "e" antigen (HBeAg), and the core protein which forms the viral nucleocapsid. However, the replication phenotype of the -1G mutant HBV is unknown. An in vitro cell culture model in which hepatoma cells were transiently transfected with infectious cDNAs was used to show that the -1G mutant HBV is incapable of autonomous replication and, as expected, replication was restored to wild-type (wt) levels by supplying HBV core protein in trans. Although the -1G mutation had no deleterious effect on intracellular HBV-DNA levels, high levels of -1G mutant HBV relative to wt HBV reduced virus secretion and HBeAg secretion relative to empty vector controls. Importantly, the -1G mutant HBV also caused intracellular retention of truncated precore protein in the endoplasmic reticulum (ER) and Golgi apparatus. Together, these effects may be contributing to the increased pathology observed in the setting of HIV/HBV co-infection.     

Strong association between genotype F and hepatitis B virus (HBV) e antigen-negative variants among HBV-infected argentinean blood donors

A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (P < 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles--T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764--were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (P < 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.     

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.     

Association of hepatitis B viral precore mutations with fulminant hepatitis B in Japan

We studied the precore DNA sequences of hepatitis B viral genomes in five patients with fulminant hepatitis B and in five with acute self-limited hepatitis B from Japan. Using the polymerase chain reaction, three to four independent HBV DNA clones from each patient were obtained and analyzed. We demonstrated that patients with fulminant hepatitis B carried HBV genomes with a G to A mutation at nucleotide positions 1898 (five of five patients; 18 of 18 clones, 100%) and 1901 (five of five patients; 12 of 18 clones, 66%) in the precore region. The first mutation results in an in-phase stop codon (TAG) in the precore open reading frame and the absence of HBeAg production. In contrast, a G to A mutation was found in 6 of 16 clones (37%) in position 1898 and in 0 of 16 clones (0%) in position 1901 from patients with acute self-limited hepatitis. We concluded that both of the precore mutations are commonly associated with fulminant hepatitis B and may contribute to the pathogenesis of fulminant hepatitis. A hypothetical model for the biological significance of these two mutations is proposed.     

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.     

Prevalence of precore-defective mutant of hepatitis B virus in HBV carriers

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant. © 1995 Wiley-Liss, Inc.     

Nucleotide sequence of hepatitis B virus isolated from subjects without serum anti-hepatitis B core antibody

The nucleotide sequences of the precore/core and X open reading frames (ORFs) of hepatitis B virus (HBV) were studied in four subjects who were serologically negative for anti-hepatitis B core antibody. These subjects were positive for serum hepatitis B surface antigen and were considered to be asymptomatic HBV carriers. Sequencing of the precore/core ORF revealed precore wild type and 3 to 8 nucleotide substitutions (replacing 0 to 2 amino acids) in the core region compared with the sequence of subtype adr. These substitutions were not considered to have changed the epitope of the core antigen, resulting in the absence of anti-HBc as determined by a conventional diagnostic kit. The X ORF showed 1 to 5 nucleotide substitutions (replacing 1 to 3 amino acids) and the structure of the X protein and the core promoter/enhancer II complex appeared to be conserved. These findings strongly suggest that the absence of serum anti-HBc is not due to mutation of the HBV DNA but to an aberrant immune reaction of the host to HBV. © 1995 Wiley-Liss, Inc.     

Detection of mutations in the enhancer 2/core promoter region of hepatitis B virus in patients with chronic hepatitis B virus infection: comparison with mutations in precore and core regions in relation to clinical status

To investigate the meaning of the mutations in the enhancer 2/core promoter (Enh2/CP) region of hepatitis B virus (HBV) during the chronic HBV infection, mutations were examined in the Enh2/ CP region (carboxyl half of X region) and their correlation with mutations in the precore and core regions in relation to the presence of chronic liver disease. The entire nucleotide sequences of the Enh2/CP region were determined by direct sequencing of the amplified products derived from 30 cases with chronic HBV infection. The results were compared to the mutations in the precore and core regions. In the Enh2/CP region, 91 generally scattered nucleotide substitutions were detected. There were 11 substitutions in the 10 asymptomatic healthy carriers (mean, 1.1/case) and 80 in the 20 chronic liver disease patients (4.0/case). The most frequent substitutions from A to T at nucleotide 1764 and from G to A at nucleotide 1766 were seen in none of the 10 asymptomatic carriers and in 14 (70%) of the 20 chronic liver disease patients. Comparisons of mutations in the precore and core regions revealed that 14 of 16 patients with mutations in the core region had the mutations in the Enh2/CP region and/or a precore stop codon mutation. These data suggest that mutations in the Enh2/CP and precore regions may affect the expression of the core and HBeAg peptides and might be involved in the pathogenesis of chronic liver disease.     

Analysis of the full-length genome of hepatitis B virus in the serum and cerebrospinal fluid of a patient with acute hepatitis B and transverse myelitis

Although many extrahepatic manifestations have been described in patients with acute or chronic hepatitis B, there are few reports about neurological disorders. We describe a 55-year-old man who contracted acute hepatitis B virus (HBV) infection and transverse myelitis. His neurological findings were gradually reduced along with the recovery from hepatitis. The cerebrospinal fluid (CSF) was revealed to be positive for HBsAg and HBV DNA. Full-length sequences of HBV in his serum and CSF were determined, and it was revealed that these two isolates had mutations at nucleotide (nt) 1762/1764 in the core promoter region and nt 1896 in the precore region. They were identical to each other except for two ambiguous codes at nt 2020 and 2631 in the CSF isolate. After cloning of the amplicons, substitutions at nt 2020 and 2631 were found in 6 (38%) of the 16 CSF clones. One clone of the 6 CSF clones had an additional substitution at nt 2119. These substitutions were not found in 16 serum clones. The presence of HBV clones unique to CSF suggests that HBV was a possible causative agent of the myelitis.     

Analysis of wild-type and e antigen-defective hepatitis B viruses during the course of a short-term corticosteroid therapy in chronic hepatitis B

Hepatitis B virus (HBV), with a G-to-A point mutation at nucleotide 83 in the precore region (mutant HBV83), accounts for most cases of hepatitis B e antigen (HBeAg)-defective HBV. However, it is still not clear how mutant HBV83 is associated with HBe seroconversion. Twenty-six HBeAgpositive patients with chronic hepatitis B who received oral prednisolone (30 mg/day) for 3 weeks were studied to clarify the prevalence of mutant HBV83 during the treatment using polymerase chain reaction with a restriction fragment length polymorphism assay. Twelve (46%) patients seroconverted to anti-HBe 1 year after treatment, whereas 14 (54%) did not. The proportion of mutant HBV83 to whole HBV remained unchanged in both groups during an acute exacerbation induced by withdrawal of corticosteroids. Among 12 anti-HBe-0seroconverted patients, five (56%) of nine patients with only wild-type HBV at baseline developed detectable levels of mutant HBV83 while all three patients with a mixed viral population of wild-type HBV and mu tant HBV83 at baseline developed a higher pro portion of mutant HBV83 one year after treat ment. In contrast, these changes were observed in only one (14%) of seven who failed to seroconvert. The results indicate that a flare-up of hepa titis precedes emergence or selection of mutant HBV83, followed by HBe seroconversion in patients with chronic hepatitis B. © 1995 WiIey-Liss, Inc.     

Sequence analysis of hepatitis B virus genomes from an infant with acute severe hepatitis and a hepatitis B e antigen-positive carrier mother

It is well known that fulminant hepatitis B can occur in infants born to hepatitis B e antigen (HBeAg)-negative hepatitis B virus (HBV) carrier mothers, whereas fulminant hepatitis and severe hepatitis are uncommon in infants born to HBeAg-positive mothers. We have encountered an infant with severe acute hepatitis B born to a HBeAg-positive mother. The aim of this study was to determine whether HBV variants contribute to the pathogenesis of fulminant hepatitis and severe hepatitis in an infant born to an HBeAg-positive mother. The nucleotide sequence of HBV genomes from the infant and his HBeAg-positive carrier mother was analyzed. All HBV isolated from the infant and his mother were subtype adr. The sequences of the cloned HBV genomes, each including a part of the X and precore/core regions, isolated from the infant were almost identical (homology of 99.1-99.9%) to those from his mother. There was no mutation in any of the 17 clones examined at nucleotides 1762 and 1764 in the core promoter, which is reported to be associated with fulminant hepatitis. A point mutation at nucleotide 1758 in the second AT-rich region of the basic core promoter was present in all clones. None of the clones had a point mutation at nucleotide 1896 of the precore region. In this study, no specific HBV variants contributing to the development of neonatal severe hepatitis were found. There is a possibility that host factors rather than viral factors play an important role in some cases of severe neonatal hepatitis B.     

Base-pair Alterations in the Epsilon-lower Stem due to a Novel Double Substitution in the Precore Gene of HBV-e Negative Variant were Recovered by Secondary Mutations

The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The destabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negative variant. We undertook to investigate presence of such compensatory mutations at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients. Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an additional G1897A substitution, presenting as a novel precore stop codon mutation (UGG-->UAA), followed by a compensatory mutation at position 1857. In the third patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also observed in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initiator which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restoration of authentic base-pairings therein, maintain the structural integrity and stability of the epsilon-hairpin. This may have a role in the enhanced selection of the HBe negative variants and persistence of HBV infection in chronic liver disease patients.     

Mutations in the Basal Core Promoter and Precore/Core Gene of Hepatitis B Virus in Patients with Chronic Active but not Acute Hepatitis B

 Around 5–10% of adults infected with hepatitis B virus (HBV) develop a chronic liver disease such as chronic active hepatitis (CAH), and it is unclear whether the clinical outcome depends solely on the immune response or whether viral factors also play a role. In this study, a search was therefore made for nucleotide mutations in the basic core promoter (BCP) and amino-acid substitutions in the precore/core region of HBV infecting patients with CAH or with acute hepatitis. The nucleotide sequences of the BCP and of the precore/core region were determined in virus from ten patients with CAH and ten with acute hepatitis. The precore/core sequences were also analysed in 14 additional patients (6 with CAH, 8 with acute hepatitis). In seven of the ten patients with CAH, five types of mutations were found in the BCP. Deletions in the precore/core region were observed in six patients. In all six patients where only the precore/core region was studied, amino-acid substitutions were present. In contrast, in the ten patients with acute hepatitis studied for BCP, a mutation was found in the BCP of one patient only. Of the 18 patients in whom the precore/core was studied, three had an amino-acid substitution in this region. The results show a clear link between CAH and both HBV BCP and precore/core region mutations, suggesting these mutations may play a role in the persistence of HBV infection.     

Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates

Summary.  We determined full-length nucleotide sequence of hepatitis B virus (HBV) genome in sera from 40 Japanese patients with HBsAg-positive hepatocellular carcinoma (HCC), in order to obtain information on HCC-specific characteristics, if any, of the HBV genome. Direct sequencing of the long distance PCR products starting from 50 μl of serum samples revealed that 95% of our isolates were of genotype C, and that mutations and deletions/insertions were very common. With respect to envelope protein genes, deletions and missense mutations were frequent in preS2, and the determinant a domain of HBsAg was rich in “antibody-escape” mutations. Within the precore/core region, the most remarkable mutation was the replacement of proline of wild type by other amino acids at codon 130 of the core gene, which was found in 58% of our isolates, while precore-stop mutation was found in 45%. Most interestingly, however, about 90% of our isolates had mutations at nt positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter, which had been implicated in “e-suppressive” phenotype of HBV genome. G-to-A at nt 1613 and C-to-T at nt 1653 within enhancer II and T-to-C/A at nt 1753 within core promoter were also evident: 38%, 53%, and 40%, respectively. It was interesting that some of the characteristics observed in our isolates form HCC patients had been previously implicated in fulminant hepatitis and/or acute exacerbation of chronic hepatitis. Received May 18, 1998 Accepted July 18, 1998     

Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences

Although Korea is a hepatitis B virus (HBV) endemic area, relatively few full-length genome sequences are available. In particular, no comparative analysis has been performed on the full-genome sequences of different HBV quasispecies from a single Korean patient. This report describes the full-length sequences of five HBV clones (two clones with shorter PCR amplicons and three clones with longer amplicons). Large deletions, that is, 685-bp, 487-bp, and 144-bp, that might interfere with the production of normal proteins were observed in four of five clones. Double mutations in the basal core promoter (BCP) region (T1762/A1764) were detected in two clones but no precore mutations (A1896) were detected in any of the five clones. These data support previous results that genotype C, in particular Korean clones of this genotype, is prone to mutations. Two independent mechanisms, namely, the deletions of long lengths and amino acid substitutions followed by BCP double mutations might contribute to the diversity of HBV quasispecies. Considering the importance of HBV quasispecies as HBV variant sources, the distribution of HBV quasispecies in mutation prone genotype C prevalent areas like Korea, should be monitored to improve the management of chronic HBV infections and to control HBV variants that arise due to the administration of vaccine or antiviral therapy.     

Two distinct types of hepatitis B virus core promoter variants in Yemeni blood donors

Genetic variations in the basic core promoter (BCP) region of hepatitis B virus (HBV) occur during the natural history of chronic HBV infection. This study investigates the presence of basic core promoter variations in 28 asymptomatic Yemeni blood donors, correlating variations with HBeAg phenotype and viral load. The core promoter/precore and surface gene region of HBV DNA were amplified using nested PCRs and the PCR products were sequenced either directly or after cloning. HBeAg and viral load were measured when HBV DNA was detectable. Sequencing of 18 surface PCR products indicated that all were of genotype D. Two distinct types of variants were identified in the basic core promoter: substitution only (N = 14) and major deletion (N = 9). The commonest substitutions were located at nucleotide positions 1753, 1762, and 1764; 10/14 (71.4%) were associated with the precore 1896A substitution resulting in the premature stop of the precore reading frame and 6/14 (42.9%) had viral loads above 400 copies/ml. Two forms of deletion variants were found: 8 bp deletion (1763-1770) (N = 2) and a novel 12 (1746-1757) + 8 bp (1763-1770) deletion (N = 7). The deletion sequences were never associated with the precore 1896A substitution and all had viral load below 400 copies/ml with negative HBeAg phenotype. The polymorphism 1773C was found in 9/14 (64.3%) substitution sequences whereas all deletion sequences had 1773T. Two donors had mixed sequences of basic core promoter substitution and major deletions (both 8 bp and 12 + 8 bp). While the deletion variants in these two donors were similar to others found in isolation, the substitutions were of a different pattern. Further studies are required to understand the selection process behind these variants.