Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment.

Hepatitis B virus (HBV) variants which cannot express e-antigen (HBeAg) are characteristic for many viremic anti-HBe positive chronic carriers who often have particularly severe and fluctuating hepatitis. Whether such variants are selected for and are less amenable to interferon treatment is under dispute. Therefore, by DNA amplification and direct sequencing we have investigated the emergence of HBV pre-C sequence variants in nine e-antigen positive chronic carriers, all of whom seroconverted to anti-HBe or lost HBeAg during interferon treatment, and in three of whom no viral DNA was detectable after interferon treatment. In most, but not all of the patients we found newly emerging pre-C sequences in a subpopulation of the viral genomes that included silent point mutations, amino acid changes, start and stop codon and frameshift mutations. The emergence of these mutations was paralleled by a drastic decrease of viremia during treatment. The observed mutations appeared most frequently during interferon treatment. Some of the mutations appeared or disappeared late after interferon treatment concomitant with anti-HBe antibody development. The appearance or lack of mutations in the pre-C region of a subpopulation of HBV of these patients was independent of successful virus elimination. These data indicate that interferon treatment is frequently associated with the simultaneous fall in titer of viral DNA by several orders of magnitude and the emergence of novel pre-C sequences, some of them preventing HBeAg expression. However, the presumably immune-mediated selection for pre-C mutant viruses and decrease in viremia under interferon treatment appears not to be prognostic for successful or unsuccessful virus elimination.     

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.     

Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region

Although rise of anti-HBe immunity in the course of hepatitis B virus (HBV) infection is generally followed by clearance of the infectious virions, ongoing chronic liver disease with circulating virions has been repeatedly observed in a significant number of anti-HBe patients, especially in Mediterranean countries. To investigate the possible role of HBV variants, we cloned HBV DNA from the serum of three such anti-HBe cases. Comparative restriction mapping of HBV clones suggested circulation of different HBV genomes in the three cases. DNA sequencing revealed an inactive pre-C region in all 11 HBV clones derived from the three cases, either as one or two point mutations in the 3' terminus generating an in-frame TAG stop codon, or a 1 nucleotide insertion in the 5' terminus resulting in frameshift mutation. Furthermore, for one clone the complete 3182 nucleotide sequence was determined and no significant mutation was found in the remainder of the genome. We conclude that chronic hepatitis cases positive for anti-HBe are associated with HBV variants containing an inactive pre-C region and hence cannot synthesize pre-C region-derived HBeAg. This finding may provide a molecular explanation for the continued viral replication despite presence of anti-HBe immunity.     

Conservation of precore and core sequences of hepatitis B virus in chronic viral carriers

Mutations in the precore region of hepatitis B virus (HBV) have been associated with failure of expression of HBV e-antigen (HBeAg), however, the prevalence of these and other mutations in HBV carriers without overt chronic liver disease remains uncertain. Homosexual or bisexual males (n = 65) with chronic HBV infection attending The Middlesex Hospital, London were studied, of whom two had clinical evidence of chronic liver disease. HBV DMA was amplified from 62 of 65 serum samples using nested and double nested polymerase chain reaction (PCR) assays. Direct sequencing of the PCR products was employed to investigate sequence variation. HBV-DNA from all available HBeAg-negative (n = 9) and selected HBeAg-positive (n = 33) sera were sequenced in the entire precore gene, the 3′ terminal portion of the X gene (aa128–154), and the 5′ terminus of the core gene (aa18–73). Sequences were highly conserved in all regions studied. Samples from two anti-HBe-seropositive patients contained mutations in the precore region. In one, a single mutation in the first amino acid resulted in a change to leucine, which would prevent translation of this region and therefore HBeAg expression. Wild type sequences were also detected in this sample. In the other sample from a patient with overt chronic liver disease, a mutation of precore amino acid 28 changed a tryptophan residue to a stop codon which would also prevent HBeAg expression. Although few such patients were studied, precore mutations may be uncommon in anti-HBe-seropositive patients without overt chronic liver disease. Possibly such mutations are not related to HBeAg to anti-HBe seroconversion, but rather they may arise in patients who remain anti-HBe seropositive for prolonged periods and they may be causally associated with the development of chronic liver disease. © 1994 Wiley-Liss, Inc.     

Epidemiology of precore mutants of hepatitis B in the United Kingdom

A point mutation assay was used to study the codon 28 and codon 1 precore mutant status of 310 chronic hepatitis B carriers (82 HBeAg positive and 228 HBeAg negative). Fourteen of 228 (6%) of HBeAg negative carriers had high levels of serum HBV DNA. Nine of these were explained by precore variants, three by core promoter variants, and two were not explained by recognised precore changes. Nested PCR detected serum HBV DNA in 36% (82/228) of HBeAg negative carriers and 63% (52/82) of these had precore variants. Four of 82 (4%) of the HBeAg positive carriers had precore variants, all as mixed mutant/wild type populations and evidence indicated that these carriers were seroconverting. Overall 23% (52/228) of HBeAg negative carriers had both serum HBV DNA and codon 1 or 28 precore mutations. A sexual transmission event from an HBeAg negative carrier with a relatively low serum HBV DNA level (10(4)-10(6) genome copies/ml) and only core promoter mutations was observed. Despite high rates of variant carriage in the antenatal sub-group perinatal transmission was not observed. The results of direct sequencing on 45 carriers validated the point mutation assay and also showed that codon 28 mutations were only seen in carriers with the genotype CCT at codon 15. For the Caucasian population a higher prevalence of codon 28 mutations (13/25 or 52%) than expected was seen. Liver biopsy data indicated that there was no link between the presence or absence of precore mutants and the severity of liver disease.     

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.     

Correlations of HBV genotypes, mutations affecting HBeAg expression and HBeAg/ anti-HBe status in HBV carriers

This study was carried out to determine the effects of hepatitis B virus genotypes, core promoter mutations (A1762G1764-->T1762A1764) as well as precore stop codon mutations (TGG-->TAG) on HBeAg expression and HBeAg/ anti-HBe status. Study was also performed on the effects of codon 15 variants (C1858/ T1858) on the predisposition of precore stop codon mutations (TGG-->TAG). A total of 77 sera samples were analyzed. Fifty one samples were successfully genotyped of which the predominant genotype was genotype B (29/ 51, 56.9 %), followed by genotype C (16/ 51, 31.4 %). Co-infections by genotypes B and C were observed in four samples (7.8 %). To a lesser degree, genotypes D and E (2.0 % each) were also observed. For core promoter mutations, the prevalence was 68.8 % (53/ 77) for A1762G1764 wild-type and 14.3 % (11/ 77) for T1762A1764 mutant while 9.1 % (7/ 77) was co-infected by both strains. The prevalence of codon 15 variants was found to be 42.9 % (33/ 77) for T1858 variant and 16.9 % (13/ 77) for C1858 variant. No TAG mutation was found. In our study, no associations were found between genotypes (B and C) and core promoter mutations as well as codon 15 variants. Also no correlation was observed between HBeAg/ anti-HBe status with genotypes (B and C) and core promoter mutations.     

A new hepatitis B virus variant in a chronic carrier with multiple episodes of viral reactivation and acute hepatitis

To examine whether there are HBV variants not yet described which cannot express HBe due to a mutation in the pre-C region, and, if they exist, whether they cause a particular course of infection and disease, we analyzed the HBV genomes of a HBs/anti-HBe positive chronic carrier who had several episodes of acute reexacerbations of chronic hepatitis with at least two viremic phases. Direct sequencing of the precore/core and the pre-S regions of the HBV sequences of both viremic phases amplified by the polymerase chain reaction revealed that they were very similar to each other but substantially divergent from published HBV genomes. Both virus populations contained a mutation in the first nucleotide of the pre-C translation initiation codon (AUG/CUG) which prevents HBeAg expression. These data demonstrate the existence of a new HBV variant which can enter a latent phase from which it can be reactivated with acute reexacerbation of liver inflammation.     

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.     

Prevalence of precore mutants in anti-HBe-positive hepatitis B virus carriers in Germany.

Hepatitis B virus (HBV) precore mutants are associated often with highly productive infection in hepatitis B surface antigen (HBsAg) carriers lacking hepatitis B e antigen (HBeAg) but positive for anti-HBe, rendering serological identification of infectious individuals unreliable. Although considered initially to be limited mostly to the Mediterranean area, more recent studies suggest a significant presence of these mutants in northern European countries. The sequence of the precore region was determined and examined for mutations from HBV isolates of 99 German chronic HBsAg carriers positive for HBV-DNA and either HBeAg (n = 15) or anti-HBe (n = 84). In addition, clinical data of individuals carrying wild-type virus and those with precore mutants were compared. HBV precore mutants were found in more than half (44/84) of all HBeAg-negative, anti-HBe-positive virus carriers. There was no difference between carriers of wild-type and precore mutant HBV in the level of viremia or in the clinical course of chronic infection. In conclusion, HBV precore mutants are common in Germany and can therefore present a diagnostic problem for serological testing. However, precore mutants do not appear to have a detrimental effect on the course of chronic HBV 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.     

'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.     

e抗体阳性的慢性乙型肝炎病人携带乙肝病毒前C区变异的研究

某些抗－ＨＢｅ（＋）的慢性乙型肝炎病人血清仍可检出ＨＢＶＤＮＡ。本工作对９例这类病人的病毒基因组，在聚合酶链反应后直接进行序列分析，发现８例其前Ｃ区第８３位核苷酸发生Ｇ→Ａ（Ａ８３）点突变，使编码色氨酸（ＴＧＧ）的第２８个密码变异为终止密码（ＴＡＧ）。其中２例Ａ８３变异发生在ＨＢｅＡｇ→抗－ＨＢｅ血清转换过程中。分析１例肝内ＨＢＶＤＮＡ，亦与其血清有相同的Ａ８３变异。以３例抗－ＨＢｅ（＋）的慢性无症状携带者为对照，均无Ａ８３变异。本文６例为慢性活动性肝炎，３例为慢性重症肝炎。变异病毒逃避了免疫清除，可能与病变持续活动甚至重症化有关。     

Strong association between genotype F and hepatitis B virus (HBV) e antigen-negative variants among HBV-infected argentinean blood donors

A number of reports have indicated an increased risk of cirrhosis and hepatocellular carcinoma in hepatitis B virus (HBV)-infected individuals carrying HBV e antigen (HBeAg)-negative variants. Although distinct core promoter and precore mutations distributed according to geographical locality and viral genotype have been reported, epidemiological data from South America are still scarce. The prevalences of HBV genotypes and core promoter and precore polymorphisms in 75 HBeAg-negative Argentinean blood donors were surveyed. The observed frequencies of HBV genotypes were 64.0% for genotype F, 17.3% each for genotypes A and D, and 1.3% for genotype C. Genotype F strains were widely distributed and significantly more prevalent in the northern region of the country (P < 0.001). An overall high proportion of a stop codon mutation (UAG) at precore codon 28 (66.7%) was observed. Wild-type codon 28 (UGG) was present in 29.3% of the samples, and the remaining 4.0% of samples had mixed variants. The combination of A at nucleotide (nt) 1762 and G at nt 1764 of the core promoter was found in 58.7% of the samples. The variant profiles--T at nt 1762 and A at nt 1764 or A at nt 1762 and A at nt 1764--were detected in 28.0 and 1.3% of the samples, respectively. The observed core promoter polymorphisms could not be related to the ratio of HBeAg to anti-HBeAg antibody, HBV genotype, or precore codon 28 status. Nevertheless, a clear association of genotype F and a precore stop codon mutation was found (P < 0.05). In conclusion, HBV genotype F and mutant codon 28 strains predominated and were strongly associated in a geographically broad Argentinean blood donor population.     

Precore and core mutations in HBV from individuals in india with chronic infection

Ten hepatitis B surface antigen seropositive carriers (5 asymptomatic and 5 with chronic liver disease) were tested for HBeAg/anti-HBe and for HBV-DNA using the polymerase chain reaction. Five were DNA-positive, 2 with HBeAg and 3 with anti-HBe. Nucleotide sequences were determined for these 5 cases. Hepatitis B virus DNA from one cirrhotic carrier with anti-HBe had a mutation in the precore region (nucleotide position 1862) which may affect signal peptide cleavage and HBeAg synthesis. In the other 2 anti-HBe- and DNA-positive cases, a cirrhotic carrier and an asymptomatic case, there was a mutation at nucleotide position 1896 leading to a termination codon in the precore region. In all 5 patients, except for one or two missense mutations, there was no significant variation in the core region. © 1995 Wiley-Liss, Inc.     

Clinical and Virological Aspects of Blood Donors Infected with Hepatitis B Virus Genotypes B and C

Pathogenic and therapeutic differences among hepatitis B virus (HBV) genotypes have been documented. However, the association of virological characteristics with clinical differences among HBV genotypes remains unclear. We therefore studied the clinical and virological characteristics of Taiwanese volunteer blood donors infected with HBV genotypes B and C. HBV genotypes were determined in 300 candidate blood donors positive for HBV surface antigen (HBsAg), and sequences of the precore gene of the HBV genome were determined in 50 HBV e antigen (HBeAg)-positive and 50 HBeAg-negative blood donors. Of 300 HBsAg-positive blood donors, 10% had elevated serum aminotransferase levels and 27% were positive for HBeAg. HBV genotype distribution in 264 viremic carriers was as follows: B, 221 (83.7%); C, 39 (14.8%); F, 1 (0.4%); and mixed infection, 3 (1.1%). Blood donors with genotype C infection tended to have a higher frequency of HBeAg positivity and a higher serum HBV DNA level than those with genotype B infection. The frequency of precore stop codon mutation was significantly higher in HBeAg-negative blood donors than HBeAg-positive ones, irrespective of HBV genotypes. Meanwhile, only 5% of blood donors with genotype C infection had C-1858 strains. In conclusion, mixed infection of HBV genotypes indeed occurs, and genotype C has a higher serum HBV DNA level than genotype B. Precore stop codon mutation is common in HBeAg-negative HBV carriers, irrespective of HBV genotypes. In contrast, precore C-1858 strains are rarely identified in Taiwanese HBV genotype C.