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.     

乙型肝炎病毒感染时的C基因热点变异

为了解乙型肝炎病毒感染在不同病变时的Ｃ基因热点变异，对９１名感染者以限制片段长度多态性技术检测前Ｃ终２８和Ｃ区Ｌ９７点突变。在急性乙型肝炎和慢性无症状携带者几无突变、在慢性持续性肝炎中罕见，而在慢性活动性肝炎和活动性肝硬变中分别达８０％和７８％，肝细胞癌中为６１％。ＨＢｅＡｇ（＋）的慢性活动性肝炎和活动性肝硬变１６例中，混合有终２８变异１１例，抗－ＨＢｅ（＋）病例中亦有野毒株混合。Ｃ区密码９７亮氨酸变异（Ｌ９７）亦见混合。野毒株和变异株在病程中有消长，Ｃ基因的热点变异与病变活动性密切相关。第一军医大学南方医院     

Evolution of wild-type and precore mutant HBV infection after liver transplantation

Recurrence of hepatitis B virus (HBV) infection after liver transplantation is associated with varying degree of graft damage. The aim of the study was to investigate longitudinally the changes of wild-type and precore A₁₈₉₆HBV mutant viral populations after reinfection and their impact on liver graft damage. The wild-type HBV and A₁₈₉₆HBV strains were quantitated before and serially after orthotopic liver transplantation (OLT) in 14 hepatitis B surface antigen (HBsAg)-positive liver graft recipients (4 hepatitis B e antigen [HBeAg]+; 10 anti-HBe+). Before OLT, the wild-type precore HBV was present in all 4 HBeAg-positive patients and in 2/10 anti-HBe-positive patients; a mixed virus population was present in 6 patients; and A₁₈₉₆HBV mutant alone in 2 patients. After OLT, A₁₈₉₆HBV mutant appeared and gradually accumulated in 5/6 patients who had the wild-type HBV before OLT and 1 of these patients seroconverted from HBeAg to anti-HBe 52 months after transplantation. A mixed HBV population was present continuously in 6 patients before and after OLT. Of the 2 patients with A₁₈₉₆HBV only pre-OLT, the wild type appeared in one patient and the other patient retained persistently the A₁₈₉₆HBV mutant. There was no relationship between liver graft histology and the type of viral population at reinfection or at the end of follow up. Changes in the HBV population occur during follow up of recurrent hepatitis B in liver transplant recipients with frequent accumulation of precore A₁₈₉₆HBV mutants, but the type of viral population does not determine the severity of hepatitis B in the graft. J. Med. Virol. 59:5–13, 1999. © 1999 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.     

Is the course of perinatal hepatitis B virus infection influenced by genetic heterogeneity of the virus?

We studied the relations between genetic heterogeneity of pre-C region of hepatitis B virus (HBV) DNA and outcome of HBV infection in 5 infants with perinatal infection, 3 born to antihepatitis B e antigen (HBeAg), and 2 to HBeAg positive mothers. HBV infection developed in the babies at 3–4 months of age, but it resolved with seroconversion to anti-HBs in infants born to anti-HBe positive mothers, while the infection became chronic in the 2 babies born to HBeAg positive mothers. HBV-DNA extracted from the first hepatitis B surface antigen (HBsAg) positive serum sample of each baby was amplified and directly sequenced for the pre-core region. HBV-DNA sequences from 3 babies born to anti-HBe positive mothers showed at position 1896 the contemporary presence of 2 nucleotides (G + A), indicating a mixture of wild-type and "e minus"variant HBV. These findings suggest a possible co-transmission of the 2 viruses from anti-HBe positive mothers to newborns. HBV-DNA from babies born to HBeAg positive mothers showed wild-type sequences only. The results of this study suggest that the outcome of HBV infection in newborns depends not only on the host's immunocompetence and on viremia level in maternal blood, but also on heterogeneity of HBV. Transmission of mixed HBV populations appears associated with an early immunoelimination of the virus, while infection with wild-type HBV alone contributes to induction of chronicity. © 1993 Wiley-Liss, Inc.     

Perinatal transmission of the hepatitis b virus and of the hbv-associated delta agent from mothers to offspring in northern italy

We report a prospective study on infants born to hepatitis B surface antigen (HBsAg) carrier mothers to estimate the incidence of perinatal transmission of HBV and HBV-associated delta agent in Northern Italy. The risk of infection to the infant was related to the presence of the HBe antigen-antibody system, HBV-specific DNA polymerase activity and antibody to delta in maternal sera, and to the titer of anti-HBe in babies at birth. The data of this study indicate: 1. Babies born to HBsAg carrier mothers with HBeAg in serum are at extremely high risk of acquiring HBV infection and of developing a chronic carrier state, whereas those born to anti-HBe-positive mothers are at a lower (P <.01) yet consistent risk of infection. 2. HBs antigenernia is usually prolonged and symptomatic in babies born to HBeAg–positive mothers while being self-limited and asymptomatic in babies born to anti-HBe-positive mothers. 3. DNA polymerase activity in maternal serum appears to be the most sensitive marker predicting HBV transmission to the infant since it was detected in all the HBeAg-positive mothers and also in two anti-HBe-positive mothers and in one HBeAg/anti-HBe-negative mother who transmitted infection to their babies. 4. High titers of anti-HBe (up to 1:10³) do not prevent HBV infection. 5. Vertical transmission of delta infection seems to occur only in circumstances that permit perinatal transmission of HBV infection.     

Evolution of the hepatitis B virus gene during chronic infection in seven patients

We analyzed the evolution of the precore/core gene of hepatitis B virus (HBV) over a period of 6 to 11 years in seven patients with chronic HBV infection, who exhibited a variety of clinical features. Sequence analysis revealed the following results: (1) HBeAg to anti-HBe seroconversion was correlated roughly with the occurrence of precore-defective mutants, and several years appeared to be required for complete replacement of wild types by mutants; (2) core gene mutations preceded precore-defective mutations and tended to increase with time, although not cumulatively. They occurred not only during serum alanine aminotransferase (ALT) elevations but also after ALT returned to normal; (3) ALT fluctuations appeared to subside with complete replacement of the wild type by the mutant type and/or substantial accumulation of core gene mutations; (4) unexpectedly, the anti-HBe-positive “healthy” carrier was found to harbor the wild type precore gene, as did the HBeAg-positive “healthy” carrier; however, the core gene of the former evolved at a rapid rate; and (5) a partial deletion was recognized in the core gene at the onset of fatal hepatic failure in one patient. Thus, the precore/core mutation was closely related with the clinical features in the patients. © 1994 Wiley-Liss, Inc.     

Clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations affecting HBV e antigen expression in Taiwan

To assess the prevalence and clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations in Taiwan, a cohort of 200 Taiwanese chronic hepatitis B patients was analyzed. The HBV genotypes and sequences of the precore and the core promoter regions were determined in 66 asymptomatic carriers and 134 patients who had liver biopsy-verified chronic hepatitis and liver cirrhosis. The HBV e-antigen (HBeAg)-negative patients had a higher frequency of mutations at core promoter nucleotides 1753 and 1773 and precore nucleotides 1846, 1896, and 1899 than HBeAg-positive patients. Among the 200 patients, the frequencies of genotype C, T1762 and A1764, C1753, T1766 and A1768, and A1896 mutations increased and the frequencies of T or G1752, T1773, G1799, and C1858 mutations decreased with advancing liver diseases. These factors were different between those with HBeAg-positive status and those with HBeAg-negative status. Based on multiple logistic regression analysis, the risk factors of liver cirrhosis for 200 patients were the presence of T1762 and A1764 mutations (odds ratio [OR] = 11.11; 95% confidence interval [CI] = 3.91 to 31.25; P < 0.001), age > or =35 years (OR = 3.42; 95% CI = 1.33 to 8.77; P = 0.011), and genotype C (OR = 2.87; 95% CI = 1.21 to 6.81; P = 0.017). Further categorical analysis found that 62.1% of patients with genotype C, T1762 and A1764 mutations and age > or =35 years had liver cirrhosis. None of the 55 patients infected with the genotype B, A1762 and G1764 wild type and age <35 years showed liver cirrhosis. In conclusion, our data suggest that pathogenic differences between HBeAg-positive and -negative patients may exist. In Taiwan, HBV genotype C and the T1762 and A1764 mutations may play a role in HBV-related liver cirrhosis, and these could serve as molecular markers for prediction of the clinical outcomes of chronic HBV patients.     

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.     

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 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.     

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 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.     

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.     

Application of a novel, rapid, and sensitive oligonucleotide ligation assay for detection of cancer-predicting mutations in the precore and basal core promoter of hepatitis B virus

Hepatocellular carcinoma (HCC) and cirrhosis are important causes of mortality worldwide. Persistent hepatitis B virus (HBV) infection is a major cause of these diseases. Double mutations in the basal core promoter (BCP) (A1762T and G1764A) and precore (pre-C) (G1896A) regions of the virus are associated with progression to HCC. The current study is aimed at developing a simple method for screening and detecting BCP and pre-C mutations in HBV carriers. We have developed and validated an oligonucleotide ligation assay (OLA) to detect point mutations in the HBV core gene. We have applied OLA methods to samples from HBV-infected carriers recruited from the Gambia Liver Cancer Study (GLCS) comprising asymptomatic HBsAg carriers, patients with cirrhosis, and patients with HCC. We observed an 89.3% and 95.8% concordance between the OLA and DNA sequencing for BCP and pre-C mutations, respectively. OLA detected the mutations in single-strain infections and in infections with mixtures of wild-type and mutant viruses under conditions where sequencing detected only the single dominant strains. BCP mutations were detected in 75.7% of patients with advanced liver disease (cirrhosis/HCC) compared to 47.6% of asymptomatic carriers, while pre-C mutations were detected in 34.5% of advanced liver disease patients and in 47.6% of asymptomatic HBsAg carriers. There was a significant association between the presence of BCP mutations and advanced liver disease. In conclusion, OLA is a simple, economical, and reliable assay for detection of pre-C and BCP mutations. Its application can lead to improvement in diagnosis and clinical care in regions where HBV is endemic.     

The precore mutation is associated with severity of liver damage in Brazilian patients with chronic hepatitis B.

BACKGROUND: The clinical relevance of the G1896A precore mutation in chronic hepatitis B is still poorly understood. OBJECTIVES: To assess the frequency of G1896A precore mutation in Brazilian patients with chronic hepatitis B, as well as its relation to the viral genotype, serum HBV-DNA levels and liver damage. STUDY DESIGN: Fifty chronic hepatitis B patients (29 HBeAg-negative and 21 HBeAg-positive) were studied. HBV-DNA was quantified by the Amplicor HBV Monitor test and precore region and S gene were amplified and submitted to automatic sequencing. The histological activity index (HAI), degrees of hepatic fibrosis and distribution of core antigen (HBcAg) in hepatocytes were determined. RESULTS: Precore mutation occurred in 1/21 (4.8%) HBeAg-positive patients and in 17/29 (58.6%) HBeAg-negative (p < 0.0001). Genotype D was identified in 56.5%, genotype A in 41.3%, and genotype F in 2.2%. The frequency of genotypes D and A, as well as serum levels of ALT and HBV-DNA were similar in patients infected with wild type and with precore mutant. Patients infected with precore mutant presented a higher frequency of moderate/severe HAI (p: 0.03) and moderate/severe fibrosis and cirrhosis (p: 0.03) than those infected with wild type. There was no association between G1896A mutation and cytoplasmic expression of HBcAg. CONCLUSIONS: Precore mutation was frequent among Brazilian subjects with chronic hepatitis B and its presence was associated with greater severity of liver disease.     

Precore and core promoter mutations of the hepatitis B virus gene in chronic genotype C-infected children

The precore (G1896A) and core promoter (A1762T, G1764A) mutations of the hepatitis B virus gene are known to be associated with changes in immunologic phase or the progression to complicated liver disease in adults. We analyzed these mutations in chronically HBV-infected children. Serum was collected from 37 children with chronic HBV infection from March 2005 to September 2008. HBV DNA extraction and nested PCR were followed by sequencing of the PCR products. The children were 6.7 ± 4.6 yr old. All of 37 children had HBV genotype C. Of the cohort, 31 (83.8%) were HBeAg-positive and 6 (16.2%) were HBeAg-negative; the former group comprised 18 (48.6%) who were in the immune-tolerance phase (ITP) and 13 (35.2%) in the immune-clearance phase (ICP). Most of the patients had HBV DNA levels of > 1.0 × 10(8) copies/mL. In the ITP group, only 1 (5.5%) had core promoter mutations, and none had the precore mutation. In the ICP group, only 2 (15.4%) had core promoter mutations; the remaining 6 patients had HBV DNA levels of < 2.0 × 10(3) copies/mL and no core promoter/precore mutations. The very low incidence of the precore/core promoter gene mutation, in children, suggests that these mutations may be the result of life-long chronic HBV infection.