Genetic variation of hepatitis B surface antigen coding region among infants with chronic hepatitis B virus infection

Variants in the amino acid composition of the primary antibody-binding site of hepatitis B surface antigen (HBsAg) have been identified in a number of populations with chronic hepatitis B virus (HBV) infection. Direct sequencing of amplified or cloned PCR products, solid phase detection of sequence-specific PCR products (SP-PCR), and limiting dilution cloning PCR (LDC-PCR) were compared to determine their sensitivity in detecting differing concentrations of HBsAg variants. LDC-PCR had the greatest sensitivity and could detect HBsAg variants at a concentration of 0.1% of the total viral population. HBsAg variants were detected in 51% of infants with chronic HBV infection acquired after postexposure prophylaxis, and more than half of the variants were detected only by the most sensitive methods.     

Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS HBsAg assay

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.     

Prevalence of naturally occurring surface antigen variants of hepatitis B virus in Korean patients infected chronically

Although Korea is one of the endemic areas of hepatitis B virus (HBV) infection, the prevalence of naturally occurring variants in the major hydrophilic region (MHR) of the surface (S) gene of HBV has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical state, and HBeAg serostatus in a large series of Korean patients with chronic HBV infection by direct sequencing analysis of part of the S gene containing the MHR of HBV isolated from 101 chronic HBV patients (51 HBeAg-positive and 50 HBeAg-negative): 37 were asymptomatic carriers, 21 had chronic hepatitis, 20 had liver cirrhosis, and 23 had hepatocellular carcinoma (HCC). Forty-seven MHR variants (46.5%) of the 101 patients were detected, involving a total of 59 amino acid substitutions at 12 positions inside and 14 position outside the 'a' determinant, and 33 'a' determinant variants (32.7%). A total of 17 novel variants and 14 novel mutation patterns were detected. The prevalence of MHR variants in HBeAg-negative patients tended to be higher than in HBeAg-positive patients (54.0% vs.39.2%) and the prevalence of MHR variants in HCC and liver cirrhosis tended to be higher than in asymptomatic carriers (65.2% vs. 40.5% and 50.0% vs. 40.5%, respectively). In conclusion, three important findings were found in the present study. First, an unexpectedly high prevalence of naturally occurring MHR variants was found in Korean chronic patients. Second, several novel variants associated with mutations outside the 'a' determinant were detected. Finally, a higher prevalence of MHR variants was associated with HBeAg-negative serostatus and severe liver disease, particularly HCC.     

Quantitative detection of hepatitis B surface antigen by chemiluminescent microparticle immunoassay during lamivudine treatment of chronic hepatitis B virus carriers

The usefulness of fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) for monitoring serum levels of hepatitis B virus (HBV) during antiviral therapy remains unclear. Using this assay, hepatitis B surface antigen (HBsAg) was measured in 20 patients with chronic hepatitis B before and during lamivudine treatment. At the start of therapy, 12 patients had detectable hepatitis B e antigen (HBeAg) and 8 did not. The median serum HBV DNA level and HBsAg concentration (25th-75th centile) were 7.2 (6.1-7.8) log genome equivalents/ml and 3,932 (1,585-12,330) IU/ml, respectively. The HBsAg concentration was significantly higher in HBeAg positive than in HBeAg negative patients (P=0.031). There was a significant correlation between the HBsAg concentration and HBV DNA level (r=0.490, P=0.027). The HBsAg concentration negatively correlated with patient age (r=-0.395, P=0.085). After the start of lamivudine therapy, HBV DNA levels fell rapidly in all patients. Serum HBsAg concentrations also fell in most patients, but to a lesser extent. When drug-resistant variants emerged, serum HBsAg usually increased before biochemical breakthrough. Although HBV DNA was elevated persistently after the emergence of drug-resistant variants, the increase in HBsAg was transient. In some patients, the increase in HBsAg preceded the increase in HBV DNA. Monitoring of serum HBsAg concentrations with the use of Architect HBsAg QT, in addition to measurement of HBV DNA levels, is helpful for evaluating the response to lamivudine treatment and for the early detection of drug-resistant strains.     

Phylogenetic analysis of new hepatitis B virus isolates from Nigeria supports endemicity of genotype E in West Africa

Isolates of hepatitis B viruses were collected from 20 acute and chronic hepatitis patients in a highly endemic region of Nigeria. Sequencing classified the isolates to the ayw4, as they all contained the amino acid variations characteristic for that serotype. In the pre-S2 region of five isolates, three to seven amino acids were deleted, suggesting that immune escape mutations previously associated only with chronic HBV infection may be observed also in acute disease. Phylogenetic analysis of the complete pre-S2/S (large S) genes (831 nt) demonstrated that all the viruses belonged to the same genotype E. So far, no isolates of genotype E have been found in any other region of the world, including the Americas. This may suggest a relatively recent introduction of this genotype into humans and would explain the relatively low genetic diversity of viruses belonging to this genotype. One genotype E virus had been found previously in a chimpanzee, and viruses belonging to the CHIMP genotype are related to other genotype E viruses. These findings are compatible with a transmission of genotype E viruses from chimpanzees to humans.     

Hepatitis B virus clearance from serum and liver after acute hepatitis delta virus superinfection in chronic HBsAg carriers

Clinical, virological, and histological findings in four HBsAg chronic carriers who cleared HBV markers from both serum and liver following HDV superinfection are described. The patients were long-term HBsAg carriers and all were HBV-DNA/HBeAg positive. Liver biopsy, obtained from three of the patients between 5 and 15 months prior to HDV superinfection, showed chronic persistent hepatitis in two and chronic active hepatitis in one. During the follow-up of 9-19 months, the patients completely recovered from acute delta hepatitis with termination of HBsAg carriage and regression of the histological feature of chronic liver damage. These data demonstrate that sometimes HDV is able to induce a permanent inhibition of its helper virus. HDV superinfection probably enhances the immune clearance of infected cells during the replicative phase of chronic HBV infection.     

Asymptomatic hepatitis B carriers who were vaccinated at birth

The long-term persistence of immunity following universal infant immunization against hepatitis B virus (HBV) and the need for a subsequent booster dose in adolescence remain under debate. With data derived from Long'an County, Guangxi, China, we reported previously that the prevalence of hepatitis B surface antigen (HBsAg) among adults born from 1987 to 1993 increases with age, although these individuals had received a first dose of the vaccine within 24 hours of birth. Here, we sought the source of transmission by comparison of genotypes among their family members using phylogenetic analysis of complete HBV S gene sequences. For comparison, we screened 2199 vaccinated individuals aged 5 to 17 in Cang Wu County and 1592 vaccinated individuals aged 3 to 7 in Ling Shan County in Guangxi for HBsAg carriers and investigate their family members. In total, 50 asymptomatic HBsAg carriers who were vaccinated at birth and 152 family members were analyzed. The results showed that 25% (95% CI: 6.0-44.0) of the HBsAg-positive children had not acquired their HBV infection from their mothers. This phenomenon showed a trend that increases with age. Antibody escape mutations were detected in 22.9% (95% CI: 11.0-34.8) of the isolates. In conclusion, a booster dose may be necessary for adolescence who were vaccinated at birth in highly endemic countries.     

Naturally occurring MHR variants in Turkish patients infected with hepatitis B virus

Major B-cell epitopes are located at the major hydrophilic region (MHR) of hepatitis B virus (HBV) surface antigen (HBsAg). The genotypes, subtypes, and naturally occurring amino acid (aa) substitutions of MHR were analyzed in 81 Turkish adult patients (41 inactive HBsAg carriers and 40 patients with chronic hepatitis B) by direct sequencing of the S gene fragment. All the isolates were genotype D according to the phylogenetic analysis. The most common HBsAg subtype was ayw2, followed by ayw3 while one isolate specified ayw4 by encoding Leu127. MHR variants were detected in 22 of the 81 (27.2%) isolates. The prevalence was significantly higher in the chronic hepatitis B group (42.5%) compared to inactive HBsAg carriers (12.2%). Twenty-two samples had a total of 26 amino acid substitutions involving 14 positions. The majority of the patients had a single variation. Most of the amino acid substitutions were located at the HBs1 region of the MHR, while 9 of the 26 were in the classic "a" determinant (aa 124-147). When samples with "a" variants were evaluated by two different commercial HBsAg tests, only the isolate with Ser143Leu variation had a decreased reactivity in the assay using monoclonal antibodies for capture and detection. In conclusion, the findings of the study was in accordance with previous studies showing HBV genotype and subtype homogeneity (genotype D/ayw) in Turkey. Naturally occurring MHR and "a" determinant variants were common, especially among chronic hepatitis B patients. The influence of detected "a" variants on diagnostic assays was limited.     

Comprehensive analysis of the prevalence of hepatitis B virus escape mutations in the major hydrophilic region of surface antigen

Escape mutations in the major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) are reported widely worldwide; these mutations lead to diagnostic problems, emergence of vaccine-escape mutants, and hepatitis B immunoglobulin (HBIG) therapy failure. However, the prevalence of these mutations in different genotypes remains to be studied systematically. In the current study, 11,221 non-redundant hepatitis B virus (HBV) sequences of 8 genotypes (from A to H), obtained from the National Center for Biotechnology Information (NCBI), were analyzed to determine the prevalence of HBsAg escape mutations that were previously described. Eight important mutations associated with diagnostic failure, P120T, T126S, Q129H, G130N, S143L, D144A, and G145A/R, were prevalent in one or more genotypes, with the frequency of no less than 1%. With regard to escape variants that evade vaccine or immunoglobulin therapy, mutations were located mainly at positions 120, 126, 129, 130, 133, 134, 137, 140, 143, 144, and 145. The majority of such mutations showed genotypic heterogeneity, indicating the different distribution of the escape mutations. Most of the escape mutations clustered in the "a" determinant, indicating that this region was more likely to be affected by immune selection or antiviral therapy than other regions. Understanding the prevalence and heterogeneity of escape mutations could provide useful guidance for the improvement of diagnostic assays, design of new vaccines, and prevention of failure of HBIG therapy.     

Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.

Methods

Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.

Results

Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.

Conclusions

Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.     

Clinical implications of hepatitis B surface antigen quantitation in the natural history of chronic hepatitis B virus infection

BackgroundHBsAg quantitation may be useful for managing patients with hepatitis B virus (HBV) infection.ObjectivesWe explored the clinical implications of HBsAg quantitation for patients with HBsAg levels >250 IU/ml (Abbott Diagnostics).Study designTwo hundred and thirty-three HBV-infected patients comprising 29 immune tolerance cases, 49 treatment-naïve HBeAg-positive chronic hepatitis B (CHB) cases, 91 inactive HBV carrier cases, and 64 treatment-naïve HBeAg-negative CHB cases were analyzed. HBsAg was quantified by the Architect HBsAg assay (Abbott Diagnostics) after a 1:500 automated dilution.Results and conclusionsHBsAg (log 10 IU/ml) was established for immune tolerance (4.50 ± 0.43), HBeAg-positive CHB (4.17 ± 0.66), inactive HBV carrier (3.32 ± 0.44), and HBeAg-negative CHB (3.23 ± 0.40); (p = 4.92 × 10⁻³⁵). No significant difference was observed between inactive HBV carrier and HBeAg-negative CHB (p = 0.247). The proportions of HBsAg <2000 IU/ml for inactive HBV carrier and HBeAg-negative CHB were 51.6% and 59.3%, respectively (p = 0.341). Positive correlations between HBsAg and HBV DNA were observed for immune tolerance (p = 1.23 × 10⁻⁴) and HBeAg-positive CHB (p = 0.003), but not for HBeAg-negative CHB (p = 0.432). A negative correlation between HBsAg and age was observed for immune tolerance (p = 0.030), HBeAg-positive CHB (p = 0.016), and inactive HBV carrier (p = 0.001), but not in HBeAg-negative CHB (p = 0.249). No significant differences between HBsAg and ALT for HBeAg-positive (p = 0.338) or HBeAg-negative CHB (p = 0.564) were observed. For patients with HBsAg quantitation >250 IU/ml, HBsAg may reflect HBV DNA replication for HBeAg-positive cases. HBsAg is not a suitable marker for evaluating hepatitis activity and distinguishing between cases of HBeAg-negative CHB and inactive HBV carrier state.     

Hepatitis B virus S gene mutants in a patient with chronic active hepatitis with circulating Anti-HBs antibodies

An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.     

Frequency of hepatitis B virus e-minus mutants varies among patients from different areas of China

Four hundred forty-six serum samples from HBsAg-positive chronic hepatitis B patients were collected from five areas in China (eastern coastal city, Shanghai; southwestern inland city, Chengdu; mid-inland city, Wuhan; southern island city, Haikou; and northeastern city, Changchun). Precore stop codon variants (e-minus mutants) were screened using a rapid method of polymerase chain reaction (PCR) amplification of a precore and partial core gene fragment (nucleotides 1785–2172), followed by dot-blot hybridization with specific oligonucleotide probes (M0, and M1 + M2). The sequence of the M0 probe covered the distal precore region of wild-type virus (nucleotides 1887–1908), and the sequences of the M1 and M2 probes were from sequences mutated at nt. 1898, (TGG→TAG) with or without additional change at nt. 1901. A significantly lower incidence of the precore stop codon was found in anti-HBe-positive serum samples from Haikou (17.6%), whereas in other areas the percentages of this mutation in anti-HBe positive sera ranged from 47.4% to 78.9%. In HBeAg-positive samples, the rate of e-minus mutant in coexistence with wild-type virus was low in specimens from Haikou (9.5%) and Changchun (2.9%) compared to other areas in China. In contrast, coexistence of mutant and wild-type virus was frequently detected in samples from Wuhan (50.0%). J Med Virol 51:85–89, 1997 . © 1997 Wiley-Liss, Inc.     

Hepatitis B virus strains in Thailand: Genomic variants in chronic carriers

Genetic heterogeneity of the hepatitis B virus (HBV) has been shown to influence the serological pattern and clinical picture in HBV infection. Thailand has a high transmission rate of HBV, but the molecular epidemiology of HBV strains circulating in this region was hitherto unknown. In this study, the HBV strains from 34 Thai HBsAg-positive patients were investigated. In a proportion of these samples, an antigenically important region of the S gene (n = 181, and the pre-S2 and precore genes (n = 15) were sequenced after PCR amplification. Four strains had in-frame deletions of an upstream region of the pre-S2 gene, with all deletions ending at the same nucleotide. In one of three anti-HBe positive strains without a translational stop at codon 28 of the precore gene, there was a one nucleotide insertion in the precore gene. This insertion would cause a frame shift and result in a nonsense protein being expressed, thus providing one explanation for the lack of HBeAg in this patient. Several rare or unique amino acid changes in the region between residues 120 and 161 of the S protein were found. Glycine 145 was changed to alanine in one strain, and this position showed an apparent mixture of glycine and arginine in another. In total, 10 strains displayed unexpected changes that were not related to the normal variability between subtypes or genetic subgroups. It is concluded that there is considerable heterogeneity in HBV strains in Thailand and that this could have clinical and epidemiological importance in a region with high HBV transmission rates. © Wiley-Liss, Inc.     

Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B

Changes in two novel HBV serological markers, linearized hepatitis B surface antigen (HQ-HBsAg) and hepatitis B core-related antigen (HBcrAg), in the natural history of chronic hepatitis B (CHB) have not been well characterized. Serum HQ-HBsAg and HBcrAg levels of 404 Asian treatment-naïve CHB patients were analysed in a cross-sectional manner. Patients were categorized into five groups: immune tolerant (IT group, n = 52), immune clearance (IC group, n = 105), hepatitis B e antigen (HBeAg)-negative hepatitis (ENH group, n = 97), HBeAg-negative quiescent group (ENQ group, n = 95) and CHB with hepatitis B surface antigen (HBsAg) seroclearance (SC group, n = 55). HQ-HBsAg and HBcrAg were measured and correlated with HBV DNA, HBsAg, HBV genotype and clinical parameters. HQ-HBsAg showed good correlation with HBsAg, especially in the ENQ group (r = 0.874, p <0.001). Correlation of HQ-HBsAg with HBV DNA was less prominent and weakest in the ENH group (r = 0.268, p 0.008). HBcrAg correlated best with HBV DNA in the ENQ group (r = 0.537, p <0.001). In the ENQ group, 42.1% of patients had undetectable HBcrAg; this subgroup of patients, when compared with those with detectable HBcrAg, had significantly lower median HBV DNA (3.17/4.48 log IU/mL, p <0.001) and HBsAg (5.05/5.96 log mIU/mL, p <0.001) levels. Forty per cent of the SC group patients had detectable HQ-HBsAg and/or HBcrAg up to 42 months after HBsAg seroclearance. When comparing anti-HBs positivity and median time after HBsAg seroclearance in the SC group with and without detectable HQ-HBsAg/HBcrAg, there was no significant difference (22.7% and 36.4%, respectively, p 0.284, and 76.5 and 93.2 months, respectively, p 0.245). HQ-HBsAg and HBcrAg showed unique patterns of distribution throughout the five disease phases of CHB, including high detectability rates after HBsAg seroclearance, opening up different possibilities for their applicability.     

The role of the hepatitis B virus infection in patients with chronic hepatitis in argentina

Over a seven-year period, we monitored 221 patients with chronic hepatitis from two medical centers. By using the counterelectrophoresis (CEP) test to detect the presence of HBsAg and anti-HBc, or both, we established that 87.7% of them had hepatitis B infection. Serum specimens originally found negative for HBsAg by CEP were further tested by reversed passive hemagglutination (RPH), and those originally found negative for anti-HBc by CEP were further tested by radioimmunoassay (RIA). Five patients were anti-HBc-positive and HBsAg-negative. No sex predominance was observed, but HBsAg incidence increased with increasing age. The HBeAg antigen was detected in 46.8% of the 161 cases tested for it; the most frequent subtype found was adw (63.7%). The present findings indicate that HBV infection largely contributes to the development of chronic hepatitis in Argentinian patients.     

Hepatitis B virus genetic diversity in Argentina: Dissimilar genotype distribution in two different geographical regions; description of hepatitis B surface antigen variants

BACKGROUND: The hepatitis B virus (HBV) molecular epidemiological data of Argentina are still scarce, since most of the previous analyses have been performed in the Metropolitan Region. OBJECTIVES: To deepen the current molecular and epidemiological information about the geographical distribution of HBV genotypes and subgenotypes, and to describe the hepatitis B surface antigen (HBsAg) variants circulating in Argentina. STUDY DESIGN: Eighty-eight Argentine partial HBsAg sequences from both the Northern and the Metropolitan Regions of the country were analyzed along with 67 Argentine HBV sequences existing in GenBank. RESULTS: Phylogenetic and amino acid sequence analysis grouped the 88 samples as genotypes A (14.8%), D (21.6%) and F (63.6%). In the Northern Region, 44 out of the 48 sequences analyzed (91.7%) grouped as genotype F. Differently, in the Metropolitan Region, the 40 samples grouped as genotype F (30.0%), genotype D (42.5%), and genotype A (27.5%). An elevated proportion (14.8%) of the genomes presented mutations in the major hydrophilic region (MHR). CONCLUSIONS: The different genotype distribution in both Argentine regions indicates that the epidemiological landscape of HBV infection appears to be the result of the diverse human migratory movements that have given shape to the present population. Our findings show that the prevalence of HBsAg variants is quite significant among the Argentine population.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.