Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan

We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000--2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69-year-old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278-646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0-0.3% and 0-0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942-AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non-Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non-fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non-fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis.     

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.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis

BACKGROUND. A nosocomial outbreak of fulminant hepatitis B occurred in five patients in Haifa, Israel. Previous investigations identified the suspected source as a carrier of hepatitis B surface antigen who was positive for antibodies to hepatitis B e antigen and had chronic liver disease. We examined the strain of hepatitis B virus (HBV) that caused this epidemic, in order to identify specific mutations in the precore or core region. METHODS. The presence of HBV was identified by polymerase-chain-reaction amplification of viral DNA in serum from the source patient, the five patients with fulminant hepatitis B, and five controls with acute, self-limited hepatitis B. The amplified viral HBV DNA samples were then cloned and sequenced. RESULTS. Sequence analysis of viral DNA established that the same HBV mutant with two mutations in the precore region was present in the source patient and the five patients with fulminant hepatic failure. This HBV mutant had significant sequence divergence from other known HBV subtypes in the X, precore, and core regions. Cloned HBV DNA derived from a hospitalized patient who had subclinical hepatitis B at the same time as the outbreak and from four other control subjects with acute, self-limited hepatitis B all contained the wild-type sequence in the precore region. CONCLUSIONS. In the outbreak we studied, a mutant hepatitis B viral strain was transmitted from a common source to five patients who subsequently died of fulminant hepatitis B infection. Naturally occurring viral mutations hepatitis B infection. Naturally occurring viral mutations in the HBV genome may predispose the infected host to more severe liver injury.     

Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection

The enhancer 2/core promoter and the X protein regions located upstream of the precore and core regions in hepatitis B virus regulate expression of core/e antigen peptides. Mutations in the precore and core regions have been reported to be associated closely with the severity of type B hepatitis, and regions regulating expression of these peptides may also be involved in severe liver damage. Mutations were examined in regions that may be related to fatal liver diseases. Nucleotide sequences and deduced amino acid sequences from 20 patients with fatal type B hepatitis (12 with fulminant hepatitis and 8 with severe exacerbation) and 10 patients with self-limited acute hepatitis were analyzed. There were 50 nucleotide alterations in the enhancer 2/core promoter region of virus from 12 patients with fulminant hepatitis (average 4.1/case), 37 alterations in 8 patients with severe exacerbation (4.6/case), and 10 mutations in 10 cases of acute hepatitis (1.0/case). The numbers of amino acid mutations in X protein were 53 in 12 cases of fulminant hepatitis (4.4/case), 27 in 8 cases of severe exacerbation (3.3/case), and 9 in 10 cases of acute hepatitis (0.9/case). In fatal cases, approximately 50% of the nucleotide mutations were located within the region spanning nucleotides 1741-1777 (14.2% of the enhancer 2/core promoter region) and 30% of the amino acid mutations in X protein were located within the region containing codons 122-132 (7.1% of X protein). In addition to mutations in the precore and core regions, mutations in the enhancer 2/core promoter and the X protein regions may be associated with the pathogenesis of fatal B hepatitis infection.     

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.     

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.     

Influences on hepatitis B virus replication by a naturally occurring mutation in the core gene

Little is known about specific naturally occurring mutations of hepatitis B virus (HBV) and underlying mechanisms of their association with fulminant hepatitis. A HBV clone isolated from a patient with fulminant hepatitis was analyzed, and the features of the particular mutations observed around furin cleavage site in core region (A2339G/G2345A) were assessed using an in vitro replication model. The clone belonged to genotype B with precore stop codon mutation (G1896A). Replication efficiency of 1.24-fold HBV genome in Huh-7 cells was increased in the presence of A2339G. Further in vitro studies using furin inhibitor indicated that the effect of the mutation was probably associated with accumulation of the full-length core protein without cleavage by furin-like protease, suggesting that a processing of the core protein might play an important role in regulation of viral replication. In conclusion, the A2339G mutation was considered as one of the viral factors involved in high replication efficiency.     

重型乙型肝炎病人中乙型肝炎病毒C基因启动子变？…

目的 了解重型乙型肝炎（乙肝）病人血清乙肝病毒（ＨＢＶ）ＤＮＡ Ｃ基因启动子（ＣＰ）的变异。方法 对用聚合酶链反应（ＰＣＲ）法扩增的血清ＨＢＶ ＤＮＡ直接测序。结果 ７例亚急性重型肝炎病人的ＨＢＶ分离株ＣＰ区分别有２￣１２个替代变异,１例病人有１１ｂｐ的碱基插入。ＣＰ变异主要发生于ＣＰ的第１和第２个ＡＴ丰富,ｎｔｌ７６２和ｎｔｌ７６４的替代变异见于７例亚急性重型肝炎病人的４例中,是ＣＰ变异的热点,     

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe

To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection.     

Completely or nearly identical hepatitis B virus strains replicate between patients with acute or fulminant hepatitis B and their respective infectious sources

Five patients with acute hepatitis B and four with fulminant hepatitis B were selected for sequencing of the precore/core gene of the virus strains. Furthermore, identical sequencing was done with the HBV of the infectious sources, i.e., the sexual partner in eight cases and a natural child (chronic carrier) infecting the mother in one case. Of the subjects responsible for the infection, four were healthy HBV carriers, three suffered from chronic hepatitis B, and one from acute and one from fulminant hepatitis B. The nucleotide sequences of HBV from both the patients and the implicated sources of infection exhibited perfect identity of the precore region and perfect or high identity of the core region. The completely or nearly identical strain of virus seemed to proliferate successively in the patients following the transmission from the infecting individuals regardless of sequence variations and infectious status. In two cases a peculiar pattern of infection and disease was found: In one married couple the husband, during the incubation period of acute hepatitis B, infected his wife, who developed fulminant hepatitis. In another married couple, both partners ultimately developed fulminant hepatitis (the wife being the source of the infection). © 1994 Wiiey-Liss, Inc.     

Molecular analysis of hepatitis B virus genomes isolated from black African patients with fulminant hepatitis B

To investigate further the possible role of mutant hepatitis B viruses in the pathogenesis of fulminant hepatitis B, the genomic sequence of hepatitis B virus isolates from 9 South African blacks with this disease, including 5 entire genomes, was analysed. Seven of the isolates were genotype A. The mutation most often reported in patients with fulminant hepatitis B, the G1896A precore stop-codon substitution, was, as expected, not present in the genotype A isolates with the exception of one in which it was accompanied by a compensatory C1858T substitution. G1896A was, however, present in the one genotype D isolate. No other precore-defective mutants were detected. The other mutation commonly found in patients with fulminant hepatitis B, the paired A1762T, G1764A substitution in the basic core promoter, was present in only one patient and G1764A in one other. The pre-surface initiation-codon mutation documented in a number of patients with fulminant hepatitis B was not found in our isolates. An 18-amino acid deletion present in the pre-surface region of one isolate has not previously been described in fulminant hepatitis B. Variations within the surface region were mainly genotype specific and not previously described. A relatively large number of mutations were present in the middle region of the core gene in those isolates without G1896A or A1762T, G1764A mutations, although the pattern was not consistent with those in published studies. Thus, as in other published series in which the entire genome of hepatitis B virus responsible for fulminant hepatitis was sequenced, we detected many mutations in different genes, but none was common to all the reported isolates.     

Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B

A pathogenic role of precore-defective mutation in the onset of fulminant hepatitis B has been suggested. However, precore-defective mutants do not always cause fulminant hepatitis B and are not always isolated from affected patients. These findings strongly suggest the presence of some additional important mutations outside the precore region in fulminant hepatitis. In the present investigation an attempt was made to sequence the X open reading frame of hepatitis B virus DNA isolated from seven patients with fulminant hepatitis B and five patients with acute hepatitis B. The latter were used as controls. Since the X open reading frame encodes the X protein and contains the core promoter/enhancer II complex, some critical mutations may enhance or disrupt the replication and expression of hepatitis B virus DNA leading to fulminant hepatitis. A C-to-T substitution was found at nucleotide (nt) 1655, an A-to-T substitution at nt 1764 and a G-to-A substitution at nt 1766 in 4, 5 and 5 patients, respectively, out of the seven with fulminant hepatitis. These substitutions were not recognized in the patients with acute hepatitis. These mutations might change the function of the X protein and core promoter/enhancer II complex. It is suggested, therefore, that these mutations, as well as the precore-defective mutation, may play an important role in the pathogenesis of fulminant hepatitis. © Wiley-Liss, Inc.     

Virological and clinical characteristics on reactivation of occult hepatitis B in patients with hematological malignancy

The virological characteristics of hepatitis B virus (HBV) implicated in the reactivation of occult hepatitis B in patients who have received hematopoietic stem-cell transplantation or chemotherapy for the hematological malignancy are not well defined. Twenty-eight HBsAg-negative patients who received hematopoietic stem-cell transplantation and 138 HBsAg-negative patients treated for malignant lymphoma with chemotherapy including rituximab were enrolled. Three of the 28 patients (10.7%) received hematopoietic stem-cell transplantation and one of the 138 (0.72%) patients treated for malignant lymphoma with chemotherapy developed de novo HBV hepatitis. Anti-HBc was detected in four and anti-HBs in two patients. Genotype Bj was detected in two and C in two of they all possessed wild-type sequences in the core promoter region. A precore stop mutation (A1896) was detected in a patient with genotype Bj who developed fulminant hepatic failure. HBV DNA was detected in pretreatment HBsAg-negative samples in two of four patients, and the HBV genome sequence identified from sera before chemotherapy and at the time of de novo HBV hepatitis showed 100% homology. In an in vitro replication model, genotype Bj with the A1896 clone obtained from a fulminant case had a replication level much higher than clones obtained from de novo hepatitis B patients with genotype Bj or C with G1896. In conclusion, this is the first report demonstrating de novo hepatitis B from the reactivation of occult HBV infection confirmed by molecular evolutional analysis. The fulminant outcome of HBV reactivation can be associated with genotype Bj exhibiting high replication due to the A1896 mutation.     

Selection of a precore mutant after vertical transmission of different hepatitis B virus variants is correlated with fulminant hepatitis in infants

The incidence of perinatal transmission of hepatitis B virus (HBV) depends on the HBeAg/anti-HBe status of the mother. While children of HBeAg-positive mothers have a 90% probability of acquiring a chronic hepatitis B virus carrier state, babies of anti-HBe-positive mothers are more likely to develop fulminant hepatitis within the first 3 to 4 months of life. There is evidence that precore (pre-C) mutations of the HBV can be associated with fulminant hepatitis. The pre-C region was therefore examined in sera from nine infants with fulminant hepatitis after vertical transmission, one HBeAg-positive and seven anti-HBe-positive mothers by polymerase chain reaction (PCR) and direct sequence analysis. In five mother/infant pairs the virus populations were characterized in addition by analysing clones of the amplified products. All mothers were infected with two or four variants of HBV with mutations at different positions of the preC genome including position 1896, which results in a stop codon. While the precore stop codon was detected in a portion of the virus populations of the HBeAg-positive and of four anti-HBe-positive mothers the dominating viral strain was represented by the wild type virus in three. In contrast, the virus populations of all babies showed the 1896 precore variant as the prevalent virus strain during the phase of active disease. In the surviving baby only wild type sequences were detected after recovery. Subtype ayw was found in all mothers and infants and adw2 was present in three mothers and in the surviving child. The findings suggest that all mothers carried a wild type HBV population with a certain number of different HBV variants. After transmission of the mixed virus population a selection process was started in the baby. The association of subtype ayw with the precore mutations and with the fatal outcome of the hepatitis B might be the result of a directed selection of this variant with a particular advantage in the viral life cycle. © Wiley-Liss, Inc.     

Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis

Pre-core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full-length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem-loop structure, changing this element to a more stable structure. Twenty-five unique amino acid substitutions were found in each open reading frame, except for the X and pre-surface 2 genes. One was located in the pre-surface 1 gene; two were in the surface gene; three were in the pre-core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre-core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre-core/core gene, including mutations in the stem-loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis. © 1996 Wiley-Liss, Inc.     

Molecular and serological evaluation of surface antigen negative hepatitis B virus infection in blood donors from Venezuela

Surface antigen negative hepatitis B virus (HBV) infection was evaluated in Venezuela, by molecular characterization of blood samples positive for antibodies to core antigen (anti-HBc) and negative for surface antigen (HBsAg) in blood donors (residual infections). HBV DNA was found in 11/258 samples (4.3%), and was significantly associated with high levels of anti-HBc antibodies (>25 UI/ml, P < 0.05), while no correlation was found between the presence of HBV DNA and the levels of anti-HBs. Synonymous and non-synonymous mutations were found in the HBV surface region (but not vaccine escape mutants) and in the precore/core region (precore mutants in 2/7 samples and 33-45 bp deletions near the N-terminal core region in 4/19 samples). While HBV genotype F prevails among HBsAg positive samples from blood donors in Venezuela, residual infection isolates were mainly genotypes A and D. Phylogenetic analysis of viral surface and core region revealed discrepancies in genotype designation in 6/9 samples, suggesting the presence of mixed infection or recombination. In conclusion, HBV residual infection in Venezuela does not seem to be frequently observed in HBV genotype F. This type of infection is frequently associated with variants exhibiting mutations in the surface gene that might be affecting the correct recognition by commercial tests, with precore mutants and with core internal deletions. These variants do not seem to cause severe liver disease, and on the contrary, were found circulating at low viremia.     

重型肝炎时乙型肝炎病毒基因型与基本核心启动子及前C区突变关系的研究

目的探讨重型肝炎（重肝）乙型肝炎病毒（HBV）基因型与基本核心启动子（BCP）及前C区突变的关系。方法采用聚合酶链反应（PCR）-限制性片段长度多态性分析技术（PCR-RFLP）对52例重肝和52例慢性乙肝（CHB）进行HBV基因分型。采用PCR产物直接测序技术，随机对15例B型和15例C型重肝患者的BCP区和前C区进行序列测定，分析HBV基因型与BCPT1762／A1764及前C区A1896突变的关系。结果泉州地区重肝的基因型以B型为主（48．08％），其次为C型（30．77％）和B／C混合型（17．31％），无A、E、F型存在。与CHB组比较，重肝组B型检出率明显降低，而C型和BIC混合型检出率明显升高。C型重肝患者BCPT1762／A1764双突变率显著高于B型（P〈0．05），而前C区A1896突变率在B、C型感染者中差异无统计学意义（P〉0．05）。结论C型感染易引起较重肝损伤，而B／C型混合感染可能是导致重肝发生的重要原因之一。C型重肝患者BCP T1762／A1764双突变率显著高于B型。