Hepatitis B e antigen (HBeAg) to anti-hepatitis B e (anti-HBe) seroconversion occurs frequently until adolescence in Japanese children with chronic hepatitis B infection, and each episode of seroconversion is generally accompanied by a decrease in viral replication. In addition hepatitis B virus (HBV) DNA is frequently detected in the serum of anti-HBe-positive patients by PCR. 1 We established that when patients are grouped based on whether they have a history of seroconversion before the age of 6 or at age 6 or older, differences in mutant viral detection rates involving the core promoter and/or the precore (pre-C) regions become apparent. 1 The reason for this difference is likely due to differences in the HBV strain or host immune defense or both. One recent study reported that the emergence pattern of the core promoter or the pre-C region mutation differs among different genotypes in anti-HBe-positive patients. 2 The present study attempts to clarify these poorly understood relationships in children.
Materials and methods.
Eighteen anti-HBe-positive Japanese children with chronic HBV infection were examined. The final serum sample in which HBV DNA was detected by the PCR screening test was used in most cases. Patients 7, 8 and 9 were enrolled only after the seroconversion, and all three had documented cases of seroconversion before the age of 6 years. Case 12 had re-HBe antigenemia after transient seroconversion at the age of 9 years, and seroconversion reoccurred at the age of 10, with persisting anti-HBe positivity thereafter. Commercial radioimmunoassays (Dinabbott, Tokyo, Japan) were used for the detection of hepatitis B surface antigen, HBeAg and anti-HBe. None of the patients had undergone treatment with interferon or specific antiviral drugs. The route of transmission, age at seroconversion, sex, elapsed time until evaluation after seroconversion and the serum alanine aminotransferase concentrations are shown in Table 1.
Extraction of DNA from sera of patients and PCR was conducted with a thermal cycler (Perkin Elmer, Chiba, Japan) as described previously. 1 Thirty-five cycles of first round PCR and 30 cycles of second round PCR were performed. All primers for amplification of the pre-S, S, core promoter, pre-C and core regions were prepared according to the sequence of HBV reported by Kobayashi et al. 3 (GenBank Accession Number M12906) as described previously. 4 After gel electrophoresis and ethidium bromide staining, each PCR product was identified by a size comparison.
The amplified DNA fragments were purified with a QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan). The purified DNA was sequenced using a BigDye Primer Cycle Sequencing, FS Ready Reaction Kit (Applied Biosystems, Chiba, Japan) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol. In the sequencing reaction the primers for second round PCR were used as the sequencing template.
We used two methods for the determination of genotype. Genotype was determined by comparing gene size polymorphism for pre-S and core regions according to the definition by Li et al. 5 In the second method, sequences in the present cases were aligned according to homologous sequences from different genotypes. A phylogenetic tree was constructed using the neighbor joining method. 6, 7 Nucleotide sequences of genotype A (GenBank accession number X75666 and X75669), genotype B (X75660), genotype C (X75665 and X75667), genotype D (X75662 and X75668), genotype E (X75664) and genotype F (X75661 and X75663) were used for the phylogenetic tree construction.
According to the definition of Li et al., 5 gene sizes of the pre-S and core genes were 519 and 555 bases in genotype A, 519 and 549 bases in genotype B and C, respectively, and 486 and 549 bases in genotype E and D, respectively. The pre-S gene was 522 bases long in all cases (3 bases larger than that reported by Li et al.). The core gene was 549 bases long in 17 of 18 patients and 555 bases long in the remaining case (Case 1). Therefore, among the 18 cases, one (case 1) was determined to be genotype A, while all of the remaining cases were determined to be either genotype B or C.
The 110th to 156th amino acids of the S protein are referred to as an “a” determinant for all HBV. Serotypes d/y and w/r are classified according to the 122nd and 160th amino acids. 8 If the 122nd amino acid is lysine (AAA sequence) or arginine (AGA sequence), the serotype is “d” or “y,” respectively, whereas if the 160th amino acid is lysine or arginine, the serotype is “r” or “w,” respectively. In the present study, all HBV strains were classified as “dr,” aside from two that were classified as “dw” and “yr,” respectively. Furthermore the genomic classification was clearly established using the phylogenetic analysis shown in Figure 1. One HBV strain was classified as genotype A according to both the definition of Li et al. and phylogenetic analysis. The remaining 17 HBV strains were classified as genotype B or C according to the definition of Lie et al. and as genotype C according to phylogenetic analysis.
The G1762GG1764 (numbering by Okamoto et al.) sequence was considered to be the wild type, whereas the mutated core promoter sequence was T1762GA1764. 9 The presence of A1896 instead of G1896 indicates a stop codon at pre-C codon 28. 10 Table 1 shows the relationship between genotypes and mutant viral detection involving the core promoter and the pre-C regions. Serum HBV DNA was not detected soon after the seroconversion in one case with genotype A (Case 1). In contrast serum HBV DNA was detected in all cases with genotype C (Cases 2 to 18), although the time until evaluation after seroconversion was variable. Among cases with genotype C, approximately one-half had either a core promoter or pre-C region mutation. However, when patients were categorized according to whether they had a history of seroconversion before the age of 6 or at age 6 or older, the prevalence of the core promoter or pre-C region mutation was 100% (9 of 9) in the latter group, which was significantly higher than that in the former group (25%; 2 of 8) (Fisher’s exact probability test;P < 0.01).
HBV strains are classified into at least five genotypes on the basis of nucleotide sequence variation of the entire genome. 8 This classification has largely replaced the earlier serologic subtype system. Subsequently a simple typing method using size polymorphisms in the pre-S and core genes was developed by Li et al. 5 In general adult Japanese patients with chronic HBV infection have either genotype B or C and rarely genotype A or D. 8 However, in the present study 17 of the 18 (94%) patients who developed seroconversion from HBeAg to anti-HBe during childhood had genotype C.
The mutation from UG1896G sequence to UA1896G (stop codon) at the pre-C codon 28 leads to a decrease in HBeAg production. 10 There are at least two hypotheses to account for the decrease in viral replication. One proposes that T1762GA1764 mutations lead to reduced transcription of viral genomic RNA. However, the findings to date have been contradictory. 11–14 The second hypothesis states that the CCC1858 sequence of codon 15 and the UG1896G sequence of codon 28 of the pre-C region (C1858-G1896 pair) form a stable hairpin structure essential for the packaging of viral pregenomic RNA in the HBV replication process. 5, 15 The genotype A strain of HBV generally has a CCC1858 sequence at codon 15, which prevents the mutation from UG1896G sequence to UA1896G at codon 28, which in turn disrupts the hairpin structure. This may account for the high prevalence of wild-type HBV strain in the pre-C region in anti-HBe-positive patients in North America and Western Europe, where most patients show the genotype A strain. 5 In the present study one patient had genotype A with CCT1858 sequence at pre-C codon 15, whereas the sequence of pre-C codon 28 was TG1896G. In this case no serum HBV DNA was detected immediately after seroconversion.
Although interferon therapy reduces circulating virus without causing an emergence of the pre-C codon 28 mutation, 16 none of the cases in the present study was treated with interferon. Furthermore the emergence of HBV mutation increased with increasing time after seroconversion. 17 In the present study the time until evaluation (mean ± sd, 2.6 ± 3.8 years) in patients with seroconversion before the age of 6 was longer than that for patients with seroconversion at 6 years of age or older (0.9 ± 0.5 years) (not significant according to Welch’s test). These factors cannot account for the difference in the prevalence of HBV mutation in the two groups. Recent studies have reported that genotype D strain (CCT1858 sequence at pre-C codon 15) has wild-type sequences in both the core promoter and the pre-C codon 28 in anti-HBe positive patients. 2 However, in the present study the 17 genotype C cases had a CCT1858 sequence at pre-C codon 15. Therefore, the difference in the sequence patterns of the core promoter and pre-C regions in the present cases was not dependent on genotype, but rather on the age at which seroconversion from HBeAg to anti-HBe was detected in our population of Japanese children.
1. Ando T, Sugiyama K, Goto K, et al. Age at time of hepatitis B e antibody seroconversion in childhood chronic hepatitis B infection and mutant viral strain detection rates. J Pediatr Gastroenterol Nutr 1999; 29: 583–7.
2. Bläeckberg J, Kidd-Ljunggren K. Genetic differences in the hepatitis B virus
core promoter and precore sequences during seroconversion from HBeAg to anti-HBe. J Med Virol 2000; 60: 107–12.
3. Kobayashi M, Koike K. Complete nucleotide sequence of hepatitis B virus
DNA of subtype adr and its conserved gene organization. Gene 1984; 30: 227–32.
4. Sugiyama K, Goto K, Ando T. Sequence analysis of hepatitis B virus
genome isolated from serum of successfully-prophylacted infants. Hepatol Res 1998; 12: 45–9.
5. Li JS, Tong SP, Wen YM, Vitvitski L, Zhang Q, Trépo C. Hepatitis B virus
genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region. J Virol 1993; 67: 5402–10.
6. Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986; 3: 418–26.
7. Ina Y. ODEN: a program package for molecular evolutionary analysis and database search of DNA and amino acid sequences. Comput Appl Biosci 1994; 10: 11–12.
8. Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis B virus
by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988; 69: 2575–83.
9. Okamoto H, Tsuda F, Akahane Y, et al. Hepatitis B virus
with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e-antigen. J Virol 1994; 68: 8102–10.
10. Carman WF, Jacyna MR, Hadziyannis S, et al. Mutations preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 1989; 2: 588–91.
11. Moriyama K, Okamoto H, Tsuda F, Mayumi M. Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus
DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. Virology 1996; 226: 269–80.
12. Scaglioni PP, Melegari M, Wands JR. Biological properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame. Virology 1997; 233: 374–81.
13. Gunther S, Piwon N, Will H. Wild type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus
with C(1653)-T, A(1762)-T, and G(1764)-A mutations in the core promoter. J Gen Virol 1998; 79: 375–80.
14. Buckwold LJ, Horn MW, Ou JH. Mechanism of suppression of hepatitis B virus
precore RNA transcription by a frequent double mutation. J Virol 1999; 73: 1239–44.
15. Kidd AH, Kidd-Ljunggren K. A revised secondary structure model for the 3′-end of hepatitis B virus
pregenomic RNA. Nucleic Acids Res 1996; 24: 3295–301.
16. Chan HLY, Hussain M, Lok ASF. Different hepatitis B virus
genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 1999; 29: 976–84.
17. Maruyama T, Kuwata S, Koike K, et al. Precore wild-type DNA and immune complexes persist in chronic hepatitis B after seroconversion: no association between genome conversion and seroconversion. Hepatology 1998; 1998; 27: 245–53.