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.     

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.     

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.     

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.     

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.     

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.     

Hepatitis may precede the occurrence of precore region mutation in hepatitis B virus genome during infection in young carriers

To understand when the mutation with a stop codon of precore region in hepatitis B virus genome occurs, the prevalence of the mutation of viral DNA clones propagated from sera of school-age carriers was investigated with respect to hepatitis B e antigen (HBeAg)/anti-HBe and sequential changes of mutants along HBeAg seroconversion were analyzed. Of 32 carriers aged 8–18 years, 14 HBeAg(+) patients had 2.2% mutant clones, whereas 8 patients with low titer anti-HBe had a higher rate of 18.1% (P < 0.01) and the highest rate of 61.3% was found in 10 patients with high anti-HBe titer (P < 0.001). By contrast, the amount of viral DNA decresed significantly in patients with anti-HBe. Sequential analysis in six cases revealed three types of seroconversion with time difference of the emergence and increase of mutant clones. It is concluded that mutation occurs at a relatively young age and increases along time and/or HBeAg seroconversion. Hepatitis might precede or accelerate the emergence and increse of mutant population which might be predictive of sustained resolution of the disease.     

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.     

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.     

Evolution of precore/core promoter mutations in hepatitis B carriers with hepatitis B e antigen seroreversion

The evolution of precore stop codon mutation (A1896) and dinucleotide mutation (T1762/A1764) in the basic core promoter (BCP) of hepatitis B virus (HBV) genome during transient seroconversion and seroreversion of hepatitis B e antigen (HBeAg) remains unclarified. Five HBeAg-positive HBV carriers who experienced transient seroconversion followed by seroreversion of HBeAg (Group I, 3.3%) and 3 HBeAg-negative HBV carriers with documented reversion of HBeAg (Group II, 2.5%) in a prospective cohort of 272 patients with chronic hepatitis B were thus identified. The sequential changes at the precore nucleotide 1896 and BCP dinucleotide 1762/1764 were determined by polymerase chain reaction and direct sequencing. At enrollement, precore A1896 and BCP T1762/A1764 were noted in 4 (50%) and 1 (13%) of the eight patients. During a median follow-up period of 58 months (range: 31-76 months), 12 episodes of transient HBeAg seroconversion followed by seroreversion were encountered in Group I patients and 3 episodes of HBeAg seroreversion in Group II patients. Accompanying acute exacerbations were found in two-thirds of patients with either HBeAg seroconversion or seroreversion. Overall, precore nucleotide A1896 remained identical in 73% and 83% of the seroconversion and seroreversion events, respectively. BCP dinucleotide T1762/A1764 remained unchanged in 94% and 92% of the seroconversion and seroreversion events, respectively. At the end of follow-up, only one had both precore and BCP mutations. In conclusion, these data suggested that HBeAg seroreversion might be due to the lack of sustained precore and BCP mutations after HBeAg seroconversion. Although uncommon, HBeAg seroreversion can be associated with hepatitis exacerbation.     

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.     

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.     

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,     

'Hbe minus' mutants of hepatitis B virus. Molecular characterization and its relation to viral genotypes

The precore-core and S genes of HBV were directly sequenced from serum samples of 42 patients with chronic hepatitis B (16 hepatitis Be antigen [HBeAg]+and 26 anti-HBe+). Viral genotype A was identified in 12 cases, genotype D in 11 and genotype F in 19 cases. Precore mutations, mainly M1 (G1896A, stop at codon 28) were similarly found among viral genotypes A and D: seven cases (58%) and six cases (55%), respectively. The selection of M1 mutants from genotype D resulted in a more stable encapsidation signal but was less stable for genotype A precore mutants. Oddly enough, the encapsidation signal of M1 precore mutants from genotype F sequences were evenly distributed among less stable (genotype A M1 mutants) and more stable encapsidation signal (genotype D M1 mutants). This study shows that the selection of precore mutants that preclude the HBeAg expression, including the M1 mutation, does not necessarily depend on the stabilization of the encapsidation signal or the viral genotype In addition, the particular behavior of genotype F genomes at precore region is described.     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

Current status in hepatitis virus research]

Seroconversion from eAg to anti-e is frequently seen in the course of chronic hepatitis B infection. This phenomenon is closely related to mutation of the precore region; a G-to-A substitution of nucleotide position 1986 replaces tryptophan to translational stop codon. This mutant is responsible for the fulminant hepatitis or acute exacerbation of chronic hepatitis B. The hepatitis C virus shows a high rate of genome variations, especially at the envelope (E and NS1) region, where a neutralizing epitope is believed to exist. According to the sequence identity, the hepatitis C virus is divided into four subtypes. The virus appears to evolve separately in geographically different areas and the subtyping may have some clinical implications, such as in interferon treatment. Hepatitis E virus has three open reading frames and share the high nucleotide identity among strains isolated from Myanmar and China. Hepatitis E is endemic in the developing world and is not present in industrialized countries.