Peculiarities in the designations of hepatitis B virus genes, their products, and their antigenic specificities: a potential source of misunderstandings.

The nomenclature of the hepatitis B virus (HBV) genes and their products has developed stepwise, occasionally in an erratic way, creating many misunderstandings, especially among those who do not know the structure of HBV and its genome in detail. One of the most frequent misunderstandings, even presented in leading journals, is the designation of HBV "e"-antigen as envelope or early antigen. Another problem area are the so-called "pre" regions in the HBV genome present upstream of both the core and the surface genes of HBV, inadvertently suggesting that they may be a part of corresponding precursor proteins. Misnomers and misclassifications are frequent in defining the subgenotypes and serological subtypes of HBV. Even the well-established terminology for HBV surface (HBs) or HBV core (HBc) antigen deviates from the conventional virological nomenclature for viral envelopes or capsid proteins/antigens, respectively. Another matter of undesirable variability between publications is the numbering of the nucleotides and the graphical representation of genomic maps. This editorial briefly explains how the nomenclature evolved, what it really means, and suggests how it could be adapted to today's knowledge.

Genetic characterization and genotyping of hepatitis B virus (HBV) isolates from donors with an occult HBV infection.

BACKGROUND AND OBJECTIVES: Screening of Thai blood donors has resulted in the detection of donors with an occult HBV infection (OBI), where HBsAg is undetectable, but hepatitis B virus (HBV) DNA is present in serum in low concentrations. This study was designed to determine whether the occurrence of OBI in donors was linked to the HBV genotype and possibly to mutations in the surface (S) and core (C) gene regions. MATERIALS AND METHODS: Mutations in the S and C gene regions in 48 Thai donors with OBI were mapped by sequencing. Genotyping was determined with the INNO-LiPA test and by phylogenetic analysis of sequences from the S and C genes. RESULTS: The majority of OBI samples were genotype C (81·3%) with 6·3% of samples being genotype B. In addition, two genotype I isolates were identified. Mutations in the S region (100%) were found especially in loop 1 of the major hydrophilic loop (MHL) at positions I110L, T114S, T126I and S113T, whereas mutations in the C region (65%) were within the basal core promoter region (position A1762T/G1764A) and precore region (position G1896A). CONCLUSION: The majority of OBI samples were HBV genotype C, although genotype I, which is newly emerging in Thailand, was also detected. The study demonstrated that OBI was probably not associated with a particular HBV genotype or with certain mutations in the S and C gene regions. However, mutations in the C gene region which could potentially impair viral replication and HBsAg production and potentially lead to OBI were identified.

Phylogenetic analyses of HBV pre-s/s genes in mother-child pairs with long-term infection by presumed vertical transmission.

Vertical transmission from mother to child, the main route of chronic hepatitis B virus (HBV) infection in the East Asia, is considered one of the most important predictors for the response to antiviral therapies as well as its complications such as cirrhosis and hepatocellular carcinoma. Therefore, it is critical in both etiologic and prognostic aspects to confirm whether or not chronic HBV infection is acquired vertically. This study investigated whether mother-to-child infection could be proved by the phylogenetic analyses of HBV pre-S/S genes ever since several decades have elapsed in mother-child pairs with presumed vertical transmission. The pre-S and S regions of HBVs were compared and analyzed phylogenetically in a total of 36 adults (18 mother-child pairs) with chronic HBV infection. All of the isolates of HBV were genotype C and serotype adr. The divergence between mothers and offsprings was 0 to 1.5%. Phylogenetic trees revealed that 17 of 18 pairs (94%) with presumed vertical transmission were grouped into the same cluster. Vertical transmission from mother to child could be strongly suggested even in adults with a history of several decades of HBV infection using the phylogenetic analyses of pre-S and S genes.

[Heterogeneity of hepatitis B virus and diagnostic potential of modern test systems for the detection of HBsAg].

AIM: Study heterogeneity ofhepatitis B virus in adult patients with chronic hepatitis B and determination of diagnostic potential of modern test systems with the detection of HBsAg with amino acid substitutions in the main hydrophilic region (MHR). MATERIALS AND METHODS: In 27 hepatitis B virus samples isolated from patients with chronic hepatitis B virus infection living in Vladimir, nucleotide sequence ofgenome region corresponding to preS1/preS2/S genes was determined. RESULTS: In all of the 27 isolates genotype D virus presented by 3 subgenotypes D1, D2, D3 was detected in 18%, 26% and 56% respectively. Based on the distribution of nucleotide substitutions in the compared functional regions of hepatitis B virus (virus entry into the cell coding site (2875 - 2991 n.b.), pre-S2/S promoter region (2994 - 3171 n.b.), 5'-end pre-S2 and S-genes sequences (3172 - 154 n.b. and 155-455 n.b.), MHR (455 - 635 n.b.) and 3'-end S-gene sequence (636 - 835 n.b.), substitutions are mostly concentrated in the promoter region of the S2/S-genes (30.8%). HBsAg serotypes were determined in 24 of 27 cases by using the predicted amino acid sequence, and in 17 cases HBsAg belonged to ayw2 (71%) serotype and in 7 cases - to ayw3 serotype (29%). Amino acid substitutions G145A, M133I, S132T localized in the main hydrophilic region and P217L, S207N, V184A localized in the C-end of the protein C that are connected with diagnostic and vaccine escape were identified in 5 isolates. CONCLUSION: Diagnostic potential of test systems with the detection of HBsAg with known amino acid sequence of the MHR region were studied. Approximately equal potential of 6 test systems to detect HBsAg with amino acid substitutions G145A, M133I and S132T localized in the MHR region were shown.

[The panels of serums, containing various subtypes and mutant forms of HBsAg, to evaluate the diagnostics sensitivity of kits oa reagents detecting HBsAg].

The detection of HBsAg in blood serum using immune-enzyme analysis techniques decisively matters both for diagnostics of acute and chronic hepatitis B and screening of donor's blood and its components, controls of persons from risk groups of hepatitis B injection. The making of panels containing wide specter of samples of blood serums with sero-variants and mutant forms of surface antigen of hepatitis B virus widespread on the territory of the Russian Federation is necessary to control analytic and diagnostic sensitivity of test systems for detecting HBsAg. The testing of reagents kits to detect HBsAg using twi panels containing recombinant and native variants of HBsAg, demonstrated that these kits enable to detect various sero-vatriants of HBsAg (ayw2, adw2, ayw3varA, ayw3varB, adrq-) in concentration 0.1-0.01 IU/l and the so called elusive mutant forms of HBsAg of recombinant and native origin (G145R, Q129R, Q129H, Q129L, T143K, T126N, T126S, D144A, M133L, K141E and P142S). At that, the sensitivity can differ during the detection of native and recombinant mutant forms. The results testify the importance of using the panels both with recombinant and native samples containing the mutant forms of HBsAg in evaluation of sensitivity of reagents kits.

Performance evaluation of 70 hepatitis B virus (HBV) surface antigen (HBsAg) assays from around the world by a geographically diverse panel with an array of HBV genotypes and HBsAg subtypes.

BACKGROUND AND OBJECTIVES: This study was conducted by the International Consortium for Blood Safety (ICBS) to identify high-quality test kits for detection of hepatitis B virus (HBV) surface antigen (HBsAg) for the benefit of developing countries. MATERIALS AND METHODS: The 70 HBsAg test kits from around the world were evaluated comparatively for their clinical sensitivity, analytical sensitivity, sensitivity to HBV genotypes and HBsAg subtypes, and specificity using 394 (146 clinical, 48 analytical and 200 negative) ICBS Master Panel members of diverse geographical origin comprising the major HBV genotypes A-F and the HBsAg subtypes adw2,4, adr and ayw1-4. RESULTS: Seventeen HBsAg enzyme immunoassay (EIA) kits had high analytical sensitivity <0.13 IU/ml, showed 100% diagnostic sensitivity, and were even sensitive for the various HBV variants tested. An additional six test kits had high sensitivity (<0.13 IU/ml) but missed HBsAg mutants and/or showed reduced sensitivity to certain HBV genotypes. Twenty HBsAg EIA kits were in the sensitivity range of 0.13-1 IU/ml. The other eight EIAs and the 19 rapid assays had analytical sensitivities of 1 to >4 IU/ml. These assays were falsely negative for 1-4 clinical samples and 17 of these test kits showed genotype dependent sensitivity reduction. Analytical sensitivities for HBsAg of >1 IU/ml significantly reduce the length of the HBsAg positive period which renders them less reliable for detecting HBsAg in asymptomatic HBV infections. Reduced sensitivity for HBsAg with genetic diversity of HBV occurred with genotypes/subtypes D/ayw3, E/ayw4, F/adw4 and by S gene mutants. Specificity of the HBsAg assays was >or=99.5% in 57 test kits and 96.4-99.0% in the remaining test kits. CONCLUSION: Diagnostic efficacy of the evaluated HBsAg test kits differed substantially. Laboratories should therefore be aware of the analytical sensitivity for HBsAg and check for the relevant HBV variants circulating in the relevant population.

Antibody to hepatitis B virus induced by injecting antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with antibody to hepatitis B surface antigen (anti-HBs) were used to induce anti-HBs in mice. The anti-idiotype-induced anti-HBs was found to recognize the group-specific a determinant of hepatitis B surface antigen and to express an interspecies idiotype. These findings suggest that anti-idiotypes may be useful as vaccines or vaccine primers.

Overexpression and purification of PreS region of hepatitis B virus antigenic surface protein adr subtype in Escherichia coli.

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80 degrees.

[The spread of hepatitis B and C viruses and the structure of HBsAg subtypes in Kabardino-Balkaria].

A seroepidemiological study of the spread of hepatitis B and C viruses (HBV and HCV) was conducted among some population groups in Kabardino-Balkaria. The structure of HBsAg subtypes was also studied in the residents of the republic. The presence of viral hepatitis B markers among the test groups of the healthy population corresponds to the parameters of moderate activity of an epidemic process. The analysis of the immunological structure of the population leads the author to assign Kabardino-Balkaria to highly HBC endemic areas. The occupational factor is demonstrated to be actively involved in the spread of HBC and it is practically of no significance for HCV. An examination could reveal the HBsAg subtype adrq+ that is uncharacteristic of this area.

Horizontal transmission of hepatitis B virus among players of an American football team.

Eleven cases of hepatitis B virus (HBV) infection (5 cases of acute hepatitis B and 6 of subclinical infection) were detected among 65 members of our university's American football team during a period of 19 months. All tested positive for antibody to the hepatitis B surface antigen (HB(s)Ag) or core antigen (HB(c)Ag). The incidence of HBV infection among team members (20.4%) was significantly higher (P<.001) than among students who tested positive for antibody to HB(s)Ag throughout the university (1.8%). We also detected a single carrier of hepatitis B e antigen (HB(e)Ag) on the team. Analysis of HB(s)Ag subtypes in 3 of 5 players with acute hepatitis B indicated that their subtype (adr) was identical to that of the HB(e)Ag carrier. All players with acute hepatitis B belonged to the same training group, which also included the HB(e)Ag carrier. Our analysis suggests that horizontal transmission of HBV can occur even in a sports team, probably due to contact with open wounds during training.

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies.

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.

Hepatitis B surface antigen with an excess or deficiency in subtypic determinants in sera from asymptomatic carriers in Japan.

Using the solid-phase enzyme immunoassay with monoclonal antibodies, hepatitis B surface antigen (HBsAg) was subtyped in sera from 5082 asymptomatic carriers who donated blood units at regional blood centers in Japan. Among them, 5004 sera contained HBsAg of a regular subtype, i.e., adw, adr, ayw or ayr, while 74 contained HBsAg with excessive subtypic determinants, such as adyw, adyr, adwr, aywr, or adywr. The presence of subtypic determinants on the selfsame particle was ascertained by sandwiching HBsAg between two monoclonal antibodies of distinct subtypic specificities. The remaining 4 sera contained HBsAg that possessed only one subtypic determinant, such as ad, ar or aw. HBsAg particles of atypical subtypes would have been given rise to by a point mutation in the S gene involving the codons regulating subtypic specificities.

The distribution of the subtypes of HBsAg in Ethiopia.

Sera from various population groups in Ethiopia previously positive for hepatitis B surface antigen (HBsAg) have been investigated for their antigenic sub-specificities. Subtype and has been observed as the predominant subtype with 83% prevalence in the study population. The same subdeterminant of the HBsAg has been observed universally as the predominant subtype in blood donors, in patients with acute hepatitis B and in one patient who was an asymptomatic chronic carrier of HBsAg. Subtype ay was observed in three of seven patients with chronic hepatitis B.

Complete nucleotide sequences of six hepatitis B viral genomes encoding the surface antigen subtypes ayw4, adw4q-, and adrq- and their phylogenetic classification.

The complete nucleotide sequences of six hepatitis B viral (HBV) genomes were determined by dideoxy chain termination sequencing of ten overlapping nucleotide fragments obtained by the polymerase chain reaction. Four of the genomes belonged to the two genomic groups E and F of HBV which have been previously identified by us on the basis of sequence divergences within the S gene. Genomic group E encodes the HBsAg subtype ayw4, group F adw4q-. The other two genomes were of Pacific origin within group C and encoded adrq-. The relationship of these complete human HBV genomes to 21 that have been previously published, together with one chimpanzee virus and four rodent hepadnaviral genomes, was investigated by constructing a phylogenetic tree utilizing a combination of distance matrix and approximate parsimonious methods. Thereby, the previously demonstrated segregation of human HBV strains into six genomic groups was confirmed. Both of the representatives of the groups E and F were found to differ by 8.1-13.6% and by 12.8-15.5% from the genomes of the other genomic groups and by 1.5 and 3.7% from each other. Since they differed by more than 8% from the genomes in the other groups, the limit originally used to define HBV, genomic groups their status as new genomic groups was confirmed. The two Pacific group C strains were found to differ by 2.7% from each other and by 4.1 to 5.4% from other group C genomes, suggesting that they diverged early from the other group C genomes. According to both the overall similarity and the phylogenetic dendrogram the F strains formed the most divergent cluster of HBV genomes favoring the concept that they represented the original HBV strains of the New World. The next split in the dendrogram segregated the A, D, E and the chimpanzee strains from the Asian B and C strains. Information on the nucleotide sequences and their encoded products of HBV strains of different genomic groups will provide a basis to understand biological variations of the HBV infection in different parts of the world.

Hepatitis B surface antigen disappearance and hepatitis B surface antigen subtype: a prospective, long-term, follow-up study of Japanese residents of Okinawa, Japan with chronic hepatitis B virus infection.

To determine the natural course of hepatitis B surface antigen (HBsAg) disappearance in chronic hepatitis B virus (HBV) infection and the factors related to its disappearance, 946 HBsAg carriers in Okinawa, Japan were prospectively followed for up to 19 years (mean = 9.2 years). The disappearance of HBsAg, as determined by radioimmunoassay (RIA), was observed in 62 (6.6%) and the overall annual disappearance rate was 0.79%/year. Its disappearance was more frequent in 60 (7.4%) of 815 serum samples negative for hepatitis B e antigen (HBeAg) by RIA at entry compared with only two (1.5%) of 131 serum samples that were HBeAg positive by RIA at entry (P < 0.05). Stepwise logistic regression analysis showed that age and HBsAg subtype were significantly associated with HBsAg disappearance (both P < 0.05), and that carriers with subtype adr (odds ratio = 2.87) had an increased probability of clearing HBsAg compared with carriers with subtype adw. Conversely, HBeAg disappearance was earlier in those with the adw subtype than in those with adr. Hepatitis B virus DNA was not detected by the polymerase chain reaction after HBsAg disappearance in any of the 62 from whom it had disappeared. The HBsAg titer, as measured by reverse passive hemagglutination, was related to the time to its disappearance; the higher the titer, the longer the time to disappearance. These findings suggest that HBeAg negativity, a more advanced age, and low titers of HBsAg are favorable factors for HBsAg disappearance in the natural course of chronic HBV infection. Moreover, HBsAg subtype adr was a predictive factor for HBsAg disappearance, whereas subtype adw was predictive of early HBeAg disappearance.

Determination of HBsAg subtypes in different high risk populations using monoclonal antibodies.

Monoclonal antibodies with restricted specificity were used in a modified commercial enzyme immunoassay for detection of HBsAg to subtype HBsAg in sera from 122 Southeast Asian refugees entering the United States, 62 inmates of a correctional facility, and 19 homosexual men. This method was able to classify HBsAg as aywl-2, ayw3, ayw4, ayr, adw2, adw4, or adr. The HBsAg subtype was identified in 183 (90.1%) of the serum samples, but the serum HBsAg concentration was too low to determine the subtype for the 20 (9.9%) remaining samples. Among the Southeast Asian refugees, aywl-2 was demonstrated in 35 (33.0%) of the subtyped serum samples, the adw2 subtype was identified in 33 (31.1%) sera, adr was detected in 37 (34.9%) sera, and the adw 4 subtype as found in 1 (0.9%). The most common subtypes in Vietnam, Laos, and Cambodia were aywl-2, adw2, and adr, respectively. In prison inmates, the ayw3 subtype accounted for 31 (52.5%) of the subtyped serum samples, an ayw2 variant and the adw2 subtype were each found in 13 (22.0%) sera, and the aywl-2 subtype was detected in 2 (3.4%) sera. Many of these inmates admitted intravenous drug use. Among homosexual men, the adw2 subtype was identified in 16 (88.9%) of the subtyped serum samples and the ayw3 subtype was detected in 2 (11.1%) sera. This subtyping method can distinguish between most of the nine major HBsAg subtypes and can be easily performed with these monoclonal antibodies and commercially available reagents.

Characteristics of HBsAg subtypes distribution in Beijing area.

This paper reports that 300 HBsAg positives were distributed in 262 families. 14 of the 262 families had two or more HBsAg positives. In the 14 families, 7 showed the same subtype HBsAg, probably due to HBV infection from family source. Another 7 families showed HBsAg of different subtypes, definitely due to infections from the community.

Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HBsAG ad and ay subtypes.

We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.

[Type B viral hepatitis. Recent findings]. / Epatite virale di tipo B. Recenti acquisizioni.

The discovery of hepatitis B surface antigen by Blumberg in 1965 offered a specific marker that could readily be employed in the clinical, epidemiological and experimental investigation of type B viral hepatitis. It has since been followed by the continuous gathering of further knowledge concerning this disease. The morphology and immunological and biochemical features of virus B, the epidemiology and prevention of hepatitis B, and marker determination techniques are examined in an overview of the present situation.

[The antigenic e system in carriers of the surface antigen of viral hepatitis B (author's transl)]. / El sistema antigénico e en los portadores de antígeno de superficie de la hepatitis por virus B.

In 1972, Magnius and Espmark on confronting different sera containing the surface antigen of hepatitis B with each other, demonstrated a new antigenic item which they called e. In later works the presence of this new antigenic specificity was associated with a greater rate of incidence of chronic hepatic lesion and a greater contagiousness of B virus. In the present study the e system was determined in a group of HBsAG positive blood donors and in a group of patients carrying HBeAG who had been admitted to the hospital for different reasons. The results showed an elevated rate of anti-e antibodies in the asymptomatic donors, and this could be correlated with clinical and biochemical indemnity of the liver function. HBsAg carriers mainly presented renal insufficiency or hematologic disorders, probably related to a deficient immune response. Determination of the e system shows its usefulness in enabling HBsAg positive carriers to be classified according to whether they present or not present hepatic lesion. The presence of HBeAg could be correlated with hepatic lesion, while HBeAc seems to determine some type of protection on those patients who have it in their sera.