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.     

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.     

Reactivation of precore variant hepatitis B virus in a child with severe aplastic anaemia

Reactivation of hepatitis B e antigen (HBeAg) negative chronic hepatitis B virus (HBV) infection due to selection of precore variant virus is an uncommon complication of previous hepatitis B infection, and virtually unrecognised in children and adolescents. A child who had received treatment with methylprednisolone and antilymphocyte globulin for severe aplastic anaemia developed high levels of detectable HBV DNA associated with hepatitis B e antibody (anti-HBe) positivity. HBV DNA was extracted, amplified and the core and precore regions sequenced from 2 samples. A mixture of wild-type and the precore variants A(1896) and A(1899) was detected in both samples, with the wild-type predominating in the second sample. Reinfection was excluded by phylogenetic analysis using Phylip and the neighbour-joining method. Precore variant Hepatitis B virus can be transmitted to children as a primary infection, and it is important that aggressive liver disease, particularly in the presence of the anti-HBe phenotype, be investigated. Further studies are needed to determine the frequency of these variants.     

Hepatitis b e antigen and antibody: detection by radioimmunoassay in chimpanzees during experimental hepatitis b

Hepatitis B e antigen (HBeAg) and its antibody (anti-HBe) were evaluated using a sensitive radioimmunoassay (RIA) in weekly serum samples obtained from nine chimpanzees experimentally infected with hepatitis B virus (HBV). In two chimpanzees with HBV infection with detectable hepatitis B surface antigen (HBsAg) for less than five weeks, and in one chimpanzee with documented HBV infection with no detectable HBsAg, HBeAg was not detected; in all three, anti-HBe became detectable early in the infection. In six chimpanzees in which HBsAg was detected for 16 weeks or longer, HBeAg was detected early in the infection; in five, anti-HBe became detectable and HBeAg unde-tectable prior to the clearance of HBsAg. The sixth remained HBsAg-positive and HBeAg-positive for more than two years and never developed anti-HBe. These results confirm the sensitivity of this RIA and its value in predicting the course of HBV infections.     

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: Does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)-positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA-positive samples were subjected to polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) to screen mutations in the Precore, core, and the X-gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA-positive. Of the 40 HBV DNA-positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg-positive patients, as compared to HBeAg-negative patients, over 1 year. At follow-up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re-designated as DNA-negative on follow-up and had wild-type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow-up, there was evidence of spontaneous reduction in the mean viral load over the 1-year study period.     

Natural seroconversion from hepatitis Be antigen to antibody among hepatitis B virus carriers in Okinawa Islands

In the Okinawa Islands, the great majority of hepatitis B surface antigen (HBsAg) carriers have already acquired antibody to hepatitis Be antigen (anti-HBe) by the age of 30 years (preliminary cross-sectional data). To elucidate natural seroconversion from hepatitis Be antigen (HBeAg) to anti-HBe among HBsAg carriers found in the islands of Okinawa Prefecture, 34 HBeAg-positive HBsAg carriers were followed for 1-6 years with serial measurements of aminotransferase levels, HBeAg, and anti-HBe. The 34 subjects included 24 patients with chronic hepatitis (group 1) and ten asymptomatic HBsAg carriers (group 2). During the follow-up period, HBeAg disappeared from 14 subjects in group 1 with the cumulative clearance rate of HBeAg of 56.3% within the first 2 years and with 10 of the 14 subsequently developing anti-HBe. Moreover, the aminotransferases in 12 of the 14 spontaneously seroconverted fell into the normal range. The annual clearance rates of HBeAg among group 1 and group 2 were 25.6% and 9.3%, respectively. The tendency for early disappearance of HBeAg during a carrier's life time or in the course of chronic hepatitis may lead to the low death rate from hepatocellular carcinoma (HCC) particularly HCC associated with hepatitis B virus infection in this area.     

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.     

Receptors for polymerized human serum albumin on hepatitis B virus particles detected by radioimmunoassay: changes in receptor activity in serum during acute and chronic infection

Receptor sites that bind polymerized human serum albumin on hepatitis B virus (HBV) particles were evaluated in purified virus particle preparations and in HBsAg positive sera, by using solid phase radioimmunoassay. The receptor, found in previous reports to be species-specific, was inhibited neither by native human serum albumin, nor by anti-human immunoglobulins or anti-C1q sera, indicating that these host components are not involved in the reaction. Differential receptor expression on various HBsAg particles was evaluated by relating levels of HBsAg to those of polyalbumin binding activity. This ratio was lower in HBsAg particles purified from hepatitis Be antigen (HBeAg) positive serum (mean ratio: 3.5) than in those obtained from anti-HBe positive serum (mean ratio: 20.1), suggesting that HBeAg/anti-HBe status influences the expression of the receptor on virus particles. Accordingly, during acute hepatitis type B receptor activity was high in sera obtained at the onset, but rapidly decreased afterwards, particularly at the time of anti-HBe seroconversion. A similar behavior of the receptor was observed in cases of chronic HBV infection, where mean receptor counts by radioimmunoassay were 3825±4422 (mean cpm ±SD) in HBeAg positive cases and 868±1196 cpm in anti-HBe positive cases (P<0.01). Seven HBsAg chronic carriers, all initially HBeAg positive, were followed up over a period of 1 to 4 yr and in all of them a progressive decrease in receptor activity was noted, independently of HBeAg persistence. These results indicate that the expression of the receptor for polymerized human albumin on HBV particles is an early event in the natural course of acute and chronic HBV infection, being followed by a progressive reduction in receptor activity, that parallels the reduction in virus replication, often independently of HBsAg serum levels.     

Age-specific prevalence and significance of hepatitis B e antigen and antibody in chronic hepatitis B virus infection in Taiwan: a comparison among asymptomatic carriers, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma

The age-specific prevalence of hepatitis B e antigen (HBeAg) and its antibody (anti-HBe) were studied by radioimmunoassay, and compared in a large series of patients with chronic hepatitis B virus (HBV) infection, including 268 asymptomatic carriers, 389 chronic hepatitis, 114 liver cirrhosis, and 278 hepatocellular carcinoma (HCC). The prevalence of HBeAg/anti-HBe in asymptomatic carriers and patients with chronic hepatitis correlated closely with age as HBeAg prevalence decreased and anti-HBe prevalence increased with increasing age (P less than 0.0005), and is probably due to high infection rate at young age in Taiwan. The prevalence of HBeAg in patients with both cirrhosis and HCC are much significantly lower and had no correlation with age. Two peaks of age-specific prevalence of HBeAg and anti-HBe were observed in patients with HCC, implicating two patterns of HBV infection in these patients. The difference in the prevalence of HBeAg and anti-HBe might indicate that asymptomatic carriers, chronic hepatitis, liver cirrhosis, and HCC are sequential sequelae of HBV infection.     

Atypical serological profiles in hepatitis B virus infection

During hepatitis B virus (HBV) infection, at least four antigen–antibody systems are observed: HBsAg and anti-HBs; preS antigen and anti-preS antibody; HBcAg and anti-HBc; and HBeAg and anti-HBe. Through the examination of these antigen–antibody systems, hepatitis B infection is diagnosed and the course of the disorder may be observed. Although the serologic findings that allow both the diagnosis of HBV infection as well as assessing of its clinical course are already well established, the dynamics of viral proteins expression and of the antibodies production may vary during the infection natural course. This causes the HBV infection to be occasionally associated with the presence of uncommon serological profiles, which could lead to doubts in the interpretation of results or suspicion of a serological result being incorrect. This paper is dedicated to the discussion of some of these profiles and their significance.     

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.     

Presence of hepatitis B virus core promoter mutations pre-seroconversion predict persistent viral replication after HBeAg loss.

BACKGROUND: In patients with chronic hepatitis B virus (HBV) infection DNA levels do not always fall after anti-hepatitis B e (anti-HBe) seroconversion. OBJECTIVES: To follow longitudinally through HB e antigen (HBeAg) loss HBV DNA levels and core promoter/precore sequences in a cohort of 21 chronic HBV carriers. STUDY DESIGN: Treatment-na?ve HBeAg seropositive HBV carriers were monitored through HBeAg loss for between 2 and 22 years (mean 9.3). Core promoter/precore sequences, genotypes, HBV DNA levels and HBe status were determined. RESULTS: Patients were grouped into those in whom serum/plasma HBV DNA remained high after HBeAg loss (group 1, n=11; HBV DNA>5log(10)IU/ml) and those in whom HBV DNA declined (group 2, n=10). Re-appearance of HBeAg was seen in seven group 1 patients. Pre-seroconversion mutations in the core promoter region including A1762T and/or G1764A were detected more frequently in group 1 (P=0.031). Overall sequence changes at sites other than 1762/1764 were more common post-seroconversion in group 1 than group 2 patients (P=0.037). CONCLUSIONS: The presence of core promoter mutations prior to HBeAg loss identified those patients in whom HBV DNA persisted at high levels and was associated with temporary re-emergence of serum HBeAg. These patients may benefit from early anti-viral treatment.     

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe

To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection.     

Dynamics and impact of perinatal transmission of hepatitis B virus in North India

The magnitude and significance of perinatal transmission of hepatitis B virus (HBV) were assessed in infants of 8,575 women, of whom 3.7% were seropositive for HBV surface antigen (HBsAg). The e antigen of HBV (HBeAg) was found in 7.8% of these carriers, the antibody to HBeAg (anti-HBe) was found in 30.1% of them, and HBsAg alone was found in 62.1% of them. The estimated incidence of HBsAg positivity by 6 months of age in infants of carrier women was significantly (P less than .001) higher than in controls (18.6% vs 3.0%). Transmission was most frequent (87.5%) if the carrier mother was HBeAg positive and was much less so if she was positive for anti-HBe (17.5%) or for HBsAg alone (9.6%). Toward the end of infancy incidence of HBsAg positivity among offspring of carriers and among controls was not different. Most infants positive for HBsAg and HBeAg continued with the infection beyond 6 months of age. It is estimated that about one-third of the adult asymptomatic HBV carriers in India evolve directly from perinatal infection, while the majority become infected during childhood or early adulthood.     

Detection of hepatitis B virus DNA in chronic carriers by the polymerase chain reaction

The polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were positive for HBV-DNA on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for DNA-HBV on dot blot hybridisation, however DNA was detected in 30 of them with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected HBV-DNA in only one patient. These results indicate that while serological and conventional DNA hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients.     

血清乙型肝炎病毒前S1抗原检测及其与病毒复制的关系

用抗Ｓ和抗前Ｓ１单抗的双抗体夹心ＥＬＩＳＡ法检测１５０例慢性乙型肝炎患者、乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者和健康人血清中的ＨＢＶ前Ｓ１抗原，其结果和ＨＢＶＤＮＡ聚合酶链反应（ＰＣＲ）、乙型肝炎血清标志的检测结果进行比较。结果表明：前Ｓ１抗原在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性组中的检出率和相对滴度显著高于ＨＢｅＡｇ阴性组（Ｐ＜０．０１）；在ＨＢｅＡｇ阴性组中，抗－ＨＢｅ阴性人群前Ｓ１抗原的检出率和相对滴度也显著高于抗－ＨＢｅ阳性人群（Ｐ＜０．０１）。前Ｓ１抗原和ＨＢＶＤＮＡ检测结果的符合率达８０％，两者检出率的相关系数ｒ＝０．９８２６（Ｐ＜０．０１）。结论：血清前Ｓ１抗原和乙型肝炎病毒的存在关系密切。     

Assessment of former and newly developed HBV assays in a Third World setting.

Newly available HBV serological assays have not been established routinely in most underdeveloped countries. Utilizing enzyme-immune assays to determine the presence of pre-S1 antigen and anti-pre-S2, and using two conventional hybridization techniques and the PCR assay to detect HBV-DNA, we studied 30 HBsAg chronic carriers and as a reference group 10 subjects whose only HBV routine marker was anti-HBc. Seventy-nine percent of the HBeAg positive carriers showed detectable HBV-DNA by a non-radioactive slot-blotting technique. The PCR assay was more sensitive than the slot-blotting technique, detecting HBV-DNA in anti-HBe positive patients with moderate or normal ALT activity. Pre-S1 antigen was mostly related to the presence of HBsAg and anti-pre-S2 was associated with active viremic state, increased ALT activity (ranges 51 to 640 IU/L), and with self-limited HBV infection. The presence of HBV-DNA in the group with anti-HBc only was detectable solely by the PCR assay. For an underdeveloped country the addition of a PCR assay or pre-S/anti-pre-S protein tests to the current assessment procedures of HBV chronic infection should be used only in selective cases. HBeAg/anti-HBe serological evaluation and HBV-DNA detection by a non-isotopic conventional hybridization technique still remain as useful tools to screen initially for the presence of viremia in chronic HBsAg carriers. The presence of HBV-DNA in individuals with anti-HBc only suggests that anti-HBc screening should be maintained and expanded to all the blood banks of less industrialized countries where the rate of HBV infection in apparently healthy people tends to be high.