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.     

Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis

BACKGROUND. The presence of the hepatitis B e antigen (HBeAg) in serum is known to be a marker of a high degree of viral infectivity. However, fulminant hepatitis may occur in persons who are negative for HBeAg. A single point mutation has been reported to produce a stop codon in the precore region of hepatitis B virus DNA and prevent the formation of the precore protein required to make HBeAg. To determine whether a precore-mutant virus is causally related to severe liver injury, we analyzed the entire precore region in viral strains isolated from patients with fatal cases and uncomplicated cases of hepatitis B. METHODS. Serum was obtained from 9 patients with fatal hepatitis B (5 with fulminant and 4 with severe exacerbations of chronic hepatitis) and 10 patients with acute, self-limited hepatitis B. Serum samples from a sex partner implicated as the source of the virus in one case of fulminant hepatitis were also studied. The 87 nucleotides in the precore region of the hepatitis B virus were amplified by the polymerase chain reaction and then directly sequenced. RESULTS. Of the nine patients with fatal hepatitis, seven had retrievable hepatitis B DNA. In all seven there was a point mutation from G to A at nucleotide 1896 of the precore region, converting tryptophan (TGG) to a stop codon (TAG). In contrast, this mutation was not found in the 10 patients with acute, self-limited hepatitis B. The hepatitis B DNA from the implicated source contained a sequence with the stop-codon mutation that was identical to the sequence in her partner, who had fulminant hepatitis. CONCLUSIONS. The presence of a mutant viral strain is associated with and may be involved in the pathogenesis of fulminant hepatitis B and severe exacerbations of chronic hepatitis B.     

Molecular epidemiology of an outbreak of fulminant hepatitis B

A nosocomial outbreak of hepatitis B occurred among the inpatients of a hematology unit. Nine of the 11 infected patients died from fulminant hepatitis. An investigation was conducted to identify the source of infection and the route of transmission. Two clusters of nosocomial hepatitis B were identified. The hepatitis B virus (HBV) genome from serum samples of all case patients, of one HBsAg-positive patient with acute reactivation of the infection, and of eight acutely infected, unrelated cases was identified by PCR amplification of viral DNA and was entirely sequenced. Transmission was probably associated with breaks in infection control practices, which occurred as single events from common sources or through a patient-to-patient route, likely the result of shared medications or supplies. Sequence analysis evidenced close homology among the strains from the case patients and that from the patient with reactivation, who was the likely source of infection. Molecular analysis of viral isolates evidenced an accumulation of mutations in the core promoter/precore region, as well as several nucleotide substitutions throughout the genome. The sequences of all patients were compared with published sequences from fulminant and nonfulminant HBV infections.     

Properties of hepatitis B virus genome recovered from Vietnamese patients with fulminant hepatitis in comparison with those of acute hepatitis

Among the many mutations found in the hepatitis B virus (HBV) genome, some have been associated with fulminant hepatitis, as exemplified by precore-defective mutations. The aim of this study was to determine whether such mutations also are found in Vietnamese cases of fulminant hepatitis B. The full-genome nucleotide sequence of HBV in three patients with fulminant hepatitis (F-2, F-3, and F-6) and one with acute hepatitis (A-3), who were admitted to Cho Ray Hospital, Ho Chi Minh City, Vietnam was ascertained. Additionally, two patients with fulminant hepatitis (F-1 and F-7) and three with acute hepatitis (A-1, A-2, and A-5) were examined only for the precore/core region of HBV. Remarkably, the nonsense mutation at precore codon 28 (Trp82Stop) was found in four of the five patients with fulminant hepatitis, while all the acute hepatitis patients harbored wild type (one had a mixture of wild and mutant types). The missense mutations within the core region, Ile97Leu and Pro130Ile/Thr/Ser, were also remarkable in fulminant hepatitis. Only F-2 was free from these precore/core mutations, but F-2 was unique in that it possessed a chimeric genotype: it could be classified into genotype C as a whole, but its X region was of genotype B, like the other four fulminant hepatitis isolates (F-1, F-3, F-6, and F-7). The codon 41 of the X protein was Pro in all three fulminant hepatitis cases examined for this region, while it was Ser in the wild-type isolates of genotype B. Of note as negative data, the mutations C1653T and T1753M of the enhancer II (Enh II) and A1762T and G1764A of the precore/core promoter regions, once reported to be relevant to severe or fulminant hepatitis, were not found in the present cases. The results with the Vietnamese cases of fulminant hepatitis corroborated results of previous studies with respect to the mutations Trp28Stop of precore and Ile97Leu and Pro130Ile/Thr/Ser of core, but not for the mutations within Enh II and precore/core promoter region. Whether the Ser41Pro mutation in the X region of genotype B HBV is Vietnam-specific or disease-specific deserves further investigation.     

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.     

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,     

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.     

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.     

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.     

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.     

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.     

Gradual accumulation of mutations in precore core region of HBV in patients with chronic active hepatitis: Implications of clustering changes in a small region of the HBV core region

The sequence in the precore and core region of the hepatitis B virus (HBV) genome in the serum of five chronic active hepatitis patients at four different stages in each individual were studied by polymerase chain reaction and DNA sequencing to determine the prevalence and type of precore and core mutants in each chronic active hepatitis (CAH) patient. Gradual changes of the virus genome in each CAH patient in precore and core regions were identified. Except for the virus from one patient, the mutant viruses showed gradual changes of genome sequences, which resulted in the generation of stop codons at the precore and core region, causing the association of active hepatitis in each patient even in the presence of anti-HBe. Mutational hot spots in the core region, which includes a clustering of changes in a small region of 14 amino acids (codons 84–97 from the start of the core gene) were found in all patients. This region of mutational hot spots in the core might be a major target of cytotoxic T lymphocytes (CTL), which has evolved under the pressure of immune selections, and these mutants might play a important role in the pathogenesis of viral hepatitis. © 1996 Wiley-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.     

Genetic heterogeneity of the precore and the core promoter region of genotype C hepatitis B virus during lamivudine therapy

It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.     

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.     

A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease

The pathogenesis of chronic liver disease (CLD) due to persistent hepatitis B virus (HBV) infection has not been defined, but the disease activity is believed to correlate with the presence of hepatitis B e-antigen (HBeAg) antigenemia and high viremia. The molecular characterization of an HBV mutant isolated from an HBeAg-negative patient with severe CLD required amplification of the circulating HBV DNA (2 pg/ml) by the polymerase chain reaction (PCR). Direct sequencing of the nucleotides from five independent amplifications of the conserved precore region consistently revealed a G to A mutation in each of the two terminal codons of the precore region. Codon 28 was mutated from tryptophan-encoding TGG to a translational stop codon, TAG; codon 29 preceding the core initiation codon was changed from GGC to GAC. For biologic evaluation of these mutations on HBV replication and expression of HBeAg in vitro, HepG2 cells were transfected with cloned, recircularized mutant HBV DNA. The transfected cells contained subviral core particles in the cytoplasm and secreted mature HBV, without HBeAg, into the medium. The findings present the first evidence that complete HBV genomes can be amplified by PCR and are replication-competent in vitro. The data also indicate that HBeAg is not necessary for replication of HBV and furthermore suggest that HBeAg is not required for the progression of HBV-induced CLD.     

Clinical and virological characteristics associated with severe acute hepatitis B

To identify early predictors of a severe or fulminant course in patients with acute viral hepatitis B (AVH-B). One hundred and thirty-eight patients with symptomatic acute hepatitis B observed from 1999 to 2012 were enrolled. For each patient, the demographics, risk factors for the acquisition of hepatitis B virus (HBV) infection, clinical, biochemical and virological data (HBV DNA, HBV DNA sequences) were recorded and analysed. The HBV mutants in the polymerase region were sought in 110 (87%) patients by direct sequencing, and the rtM204V/I mutations also by an allele-specific PCR. AVH-B was severe in 13 (9.4%) of the 138 patients enrolled, fulminant in 6 (4.3%) and with a normal clinical course in 119. The 19 patients with severe or fulminant AVH-B more frequently than the 119 with a normal course stated intravenous drug use (63.2% versus 36.1%, p 0.04) and were HBV-DNA negative (31.6% versus 11.8%, p 0.03) and anti-hepatitis C virus (HCV) positive (57.9% versus 19.3%, p 0.0008); the prevalences of different HBV genotypes and of the rtM204V/I mutant were similar in these three forms of AVH-B. A multivariate logistic regression analysis identified a pre-existing HCV chronic infection as the only factor independently associated with a severe or fulminant clinical course of AVH-B (OR 4.89, 95% CI 1.5–15.94, p 0.01). A pre-existing HCV chronic infection was identified as the only factor independently associated with a severe clinical presentation of acute hepatitis B, an association most probably due to the combination of the liver lesions caused by acute hepatitis B and the pre-existing histological abnormalities related to HCV chronic infection.