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Upon phylogenetic analysis of a partial S gene sequence [396 nucleotides (nt)], 928 hepatitis B virus (HBV) strains obtained from 899 viremic subjects in 28 major cities on 15 islands of Indonesia in 1989-2007 segregated into four HBV genotypes. Genotype B was predominant (66%), followed by genotype C (26%), genotype D (7%), and genotype A (0.8%). Comparative and phylogenetic analyses of the 396-nt S gene sequence of 928 HBV isolates and whole genomic sequences of 25 selected HBV isolates revealed a total of 14 subgenotypes within genotypes A-D: two (A1 and A2) in genotype A (HBV/A), five (B2, B3, B5, B7, and a novel subgenotype, tentatively designated B8) in HBV/B, five (C1, C2, C5, C6, and another novel subgenotype, C7) in HBV/C, and two (D1 and D3) in HBV/D. The distribution of HBV genotypes/subgenotypes, including B8 and C7, seems to be associated with ethnological origins in Indonesia.

To investigate the prevalence of hepatitis B virus (HBV) genotypes and characteristics of HBV isolates among Japanese patients infected with human immunodeficiency virus type 1 (HIV), serum samples collected between September 1990 and March 2002 from 471 HIV-infected patients (age, 38.8 +/- 11.4 [mean +/- standard deviation] years; male, 90%) were tested for hepatitis B surface antigen (HBsAg) and HBV DNA. Positivity for HBsAg and HBV DNA was seen in 42 patients (8.9%), 41 of whom had contracted HIV infection through sexual activity and 1 had hemophilia. Genotypes of HBV were determined by comparative and phylogenetic analyses of the S gene sequence (396 nucleotides [nt]). The distribution of HBV genotypes among the 42 HBV-viremic patients was: A (50%), B (5%), C (24%), D (5%), E (2%), H (10%), A plus D (2%), A plus G (2%). The hemophilia patient had HBV genotype D. Genotypes E, G, and H which had not been reported in Japan, were found in one patient each who had traveled to Zambia, the US, and South America, respectively. Genotypes A and D, which are rare in Japan, were found in patients who had no history of traveling abroad. The entire genome of the HB-JI411 (genotype E [3,212 nt]), HB-JI444G (genotype G [3,248 nt]), and HB-JI260 (genotype H [3,218 nt]) isolates had the highest identity of 98.3%, 99.9%, and 98.5%, respectively, with reported HBV isolates of the same genotype. Most Japanese patients coinfected with HIV and HBV had HBV genotypes that are found rarely or had not been reported in Japan., (Copyright 2005 Wiley-Liss, Inc.)

The prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.

An ELISA was developed for serological determination of the six genotypes of hepatitis B virus (HBV) designated A, B, C, D, E, and F. Monoclonal antibodies were raised against genotype-specific epitopes in the preS2-region product, and labeled with horseradish peroxidase. Hepatitis B surface antigen (HBsAg) in sera was captured by immobilized antibodies against the common determinant, and evaluated for reactivity with genotype-specific monoclonal antibodies labeled with the enzyme. Serological genotyping was in complete accord with genotypes determined by S-gene sequences in a panel of 68 sera containing HBV/HBsAg of different genotypes. Of 514 sera with HBsAg from Japan, 507 (98.6%) were genotyped serologically: genotype A was identified in 24 (4.7%); B in 196 (38.1%); C in 282 (54.9%); D in 2 (0.4%); and F in 3 (0.6%). There were no sera containing HBV of genotype E. Likewise, 425 of 446 (95.3%) sera with HBsAg from Brazil, China, India, Indonesia, Kenya, Korea, Nepal, Papua New Guinea, the Philippines, and Thailand were classified into A (25.6%), B (24.2%), C (33.9%), and D (11.7%) genotypes; there were no sera with HBsAg of genotype E or F among them. Some sera unclassifiable by ELISA revealed mixed infection with HBV of distinct genotypes, or contained HBsAg deprived of genotype-specific epitopes by point mutations. The ELISA would be useful for large-scale surveys, because it allows serological detection of HBV genotypes without sequencing nucleotides.

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of hepatitis B virus (HBV) core protein (p21c) using monoclonal antibody (mAb) directed to a phosphorylated C-terminal amino acid sequence that is not present in hepatitis B e antigen (HBeAg). HBV virions in the test serum were precipitated with horse polyclonal antibody to hepatitis B surface antigen (HBsAg), dissolved with Tween 20 and NaOH, and then neutralized. HBV core protein (p21c), released from HBV cores by this procedure, was sandwiched between immobilized mAb C33 directed to amino acids (aa) 133-140 of the core protein, fixed on a solid support and labeled mAb T2212 that recognizes aa 165-175, only when they are phosphorylated. The method was applied for the detection of phosphorylated p21c in sera from symptom-free carriers and patients with chronic hepatitis. The results indicated a higher extent of phosphorylation in p21c of HBV cores from symptom-free carriers than hepatitis patients.

Sixty-eight consecutive patients with chronic hepatitis B received 702 million units of recombinant interferon-alpha 2a. Of the 24 patients negative for hepatitis B e antigen (HBeAg) in serum, the normalization of serum transaminase occurred in 14 (58%) at the completion of interferon therapy and in 13 (54%) at 12 months thereafter; it was normalized in 17 (39%) and 13 (30%), respectively, of the 44 HBeAg-positive patients. Of the HBeAg-negative patients, hepatitis B virus DNA was cleared from serum in six (25%) at the completion and in one (4%) at 12 months thereafter, in contrast to only one (2%, p < 0.05) and none of the HBeAg-positive patients, respectively. The 1896th nucleotide of G (G1896) for codon 28 for tryptophan or A (A1896) for the stop codon 28 in the precore region was determined by restriction fragment length polymorphism. The ten HBeAg-negative patients with A1896 only in the precore region had lower pretreatment levels of viral markers, which decreased more rapidly and extensively after interferon than in the 14 HBeAg-negative patients with a mixture of G1896 and A1896 or in the 44 HBeAg-positive patients. These results indicate that patients with HBeAg-negative chronic hepatitis B may respond better to interferon than HBeAg-positive patients, and that the precore mutant with the stop codon 28 may be sensitive to interferon.

Hepatitis B virus variants harboring nucleotide alterations in the preC-C promoter have been detected in fulminant hepatitis B as well as in HBeAg-seronegative persistent infection. However, it has not been demonstrated that variants with nucleotide alterations in the preC-C promoter cause various disease states. We replaced the preC-C promoter region of a wild-type genome with the most frequent naturally occurring mutated form and introduced it into HepG2 cells. The mutant with coexisting A1762T and G1764A substitutions produced less than one-fifth of the wild-type level of HBeAg. Conversely, the mutant generated 2.4 times more core particle antigen and showed a high-replicator phenotype. RNase protection and quantitative 5' RACE showed a 16- to 32-fold reduction of preC transcripts and a 4-fold induction of C transcripts of the mutant compared to wild-type. The preC transcript of the mutant had a more heterogeneous 5' end than that of the wildtype. However, the mutations did not alter the initiation sites of C transcription. When the promoter region was cloned into CAT plasmids, the mutations had dual effects on preC and C promoter activities, decreasing and increasing them, respectively. These results suggest that these mutations are responsible for the reduced HBeAg production as well as the enhanced replication and core production. Analysis of revertants with either single point mutation showed that T at 1762 is critical for the mutant phenotype.

Objectives: Mutations in the precore region are not always detected in hepatitis B virus (HBV) from patients with chronic hepatitis B who respond to interferon and lose hepatitis B e antigen (HBeAg) from serum. The other mutations may also be responsible for the loss of HBeAg and response., Methods: Forty six consecutive patients with HBeAg-positive chronic hepatitis B received recombinant-alpha 2 a interferon (total dose: 702 MU). The mutation for stop codon 28 in the precore region was determined by restriction fragment length polymorphism, and mutations in the core promoter were searched for in five HBV DNA clones propagated from each patient., Results: HBeAg was cleared at 6 months after interferon in 11 (61%) of 18 patients with the precore mutation and in 12 (43%) of 28 without it. Of these 28 patients, 19 with mutations in the core promoter in all five HBV DNA clones lost HBeAg more frequently than the remaining nine who had at least one clone among the five that lacked such mutations (58 vs 11%, p < 0.05)., Conclusions: HBeAg-positive patients infected with HBV variants having mutations for an HBeAg-negative phenotype would respond better to interferon by clearing HBeAg from serum. Such mutations may not necessarily be in the precore region but also in the core promoter, which would interfere with the synthesis and secretion of HBeAg either at the translation or transcription level.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124-147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130-132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of lle or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

From September 9 to October 3, 1994, five patients on maintenance hemodialysis in a dialysis unit in Tokyo contracted hepatitis B virus (HBV) infection successively, and four of them died of fulminant hepatitis. The unit treated 181 patients three times a week on eight shifts, and all five afflicted patients were on the same shift along with 27 other patients. HBV DNA clones from the hepatitis patients had a point mutation converting codon 28 in the precore region to a stop codon, which aborts the synthesis and secretion of hepatitis B e antigen, and showed a sequence similarity of > 99.5% within 645 base pairs covering the X gene and precore region. There were two HBV carriers with antibody to hepatitis B e antigen who were receiving hemodialysis on the same shift. HBV DNA clones from one of them had the stop codon 28 in the precore region, and a sequence similarity of > 99.7% to those from the five patients. Based on these results, it was deduced that the fulminant HBV strain was transmitted from the carrier to five patients, and resulted in the death of four. The outbreak indicates that immunocompromised hosts like hemodialysis patients can develop fulminant hepatitis B if and when they are infected with extremely virulent HBV strains.

Objective: Fulminant hepatitis B can be induced by hepatitis B virus (HBV) strains with mutations in the precore region that cannot encode hepatitis B e antigen (HBeAg). Such mutations are rarely seen in HBV DNA clones from patients with fulminant hepatitis B in the United States and France. Thus, the other mutations in HBV strains causing fulminant hepatitis B need to be identified., Design: Retrospective clinical, serologic, and molecular biological studies of patients with fulminant hepatitis B., Setting: University and city hospitals in Japan., Patients: 43 patients with fulminant hepatitis B., Measurements: The precore region coding for a part of the HBeAg precursor and the core promoter regulating the transcription of precore messenger RNA were sequenced in HBV DNA clones., Results: A point mutation from G to A at nucleotide 1896 in the precore region was detected in 519 (98%) of 529 HBV DNA clones from 38 patients. Two point mutations in the core promoter, from A to T at nucleotide 1762 and from G to A at nucleotide 1764, were detected in all 130 clones from the remaining 5 patients, who did not have mutations in the precore region, and in 20 (63%) of 32 clones from a patient with chronic hepatitis B who had transmitted HBV to 1 of these other 5 patients. Mutations in the core promoter were also detected in clones from 26 (68%) of the 38 patients with the precore mutation at nucleotide 1896. Neither HBeAg nor antibody to HBeAg was detected in 37 (90%) of the 41 patients tested., Conclusions: In Japan, fulminant hepatitis B is closely associated with HBV strains that do not produce HBeAg because of mutations in the precore region, which affect translation of HBeAg, or because of mutations in the core promoter, which affect transcription of the HBeAg coding region.

Hepatitis B virus (HBV) DNA clones were propagated from 57 carriers with antibody to hepatitis B e antigen (HBeAg) and sequenced within nucleotides (nt) 1685 to 1926 including the core promoter (nt 1742 to 1849) and the pre-C region (nt 1814 to 1900). Mutations in the core promoter or those in the pre-C region, or both, were detected in 328 (97.9%) of 335 clones from them. Five carriers were infected with HBV mutants with mutations in the core promoter alone, while 20 carriers were infected only with those in the pre-C region to abort the translation of HBeAg precursor; the remaining 32 carriers were infected with HBV mutants with mutations in both the core promoter and pre-C region. Some carriers infected with HBV with mutations in the core promoter exclusively had high HBV DNA titers, comparable with those in carriers infected with wild-type HBV, thereby indicating that such mutations would not affect the transcription of the HBV pregenome extensively. Two point mutations in the core promoter, from A to T at nt 1762 and from G to A at nt 1764, were most prevalent. The other mutations included a point mutation at either of the two nucleotides and their deletion. All of these mutations involved the TTAAA sequence (nt 1758 to 1762) at 28 bp upstream of the initiation site for shorter pre-C mRNAs (nt 1790 +/- 1). The ATAAATT sequence (nt 1789 to 1795) at 23 bp upstream of the initiation site for the pregenome RNA (nt 1818), however, remained intact in all 335 HBV DNA clones. HBV mutants with mutations in the core promoter, unaccompanied by pre-C mutations, prevailed and replaced wild-type HBV in two carriers as they seroconverted from HBeAg to the corresponding antibody. These results indicate that HBV mutants with an HBeAg- phenotype would be generated by mutations in the core promoter which might abort the transcription of pre-C mRNA but do not seriously affect that of pregenome RNA.

The entire nucleotide sequence was determined for eight hepatitis B virus (HBV) genomes from three symptom-free carriers, two patients with chronic persistent hepatitis and one patient with chronic active hepatitis, who were positive for antibody to hepatitis B e antigen (HBeAg). The two patients with chronic persistent hepatitis were tested again after they developed chronic active or fulminant hepatitis, making a total of eight samples. Six had a point mutation in the preC region prohibiting the encoding of HBeAg precursor, while the remaining two had a deletion of 8 or 21 nucleotides within the X gene upstream of the preC transcription initiation sites which would affect the X gene and the putative preC/C promoter. Most genomes from the three symptom-free carriers and the two patients with chronic persistent hepatitis, with HBV DNA levels of 10(2)-10(3)/ml, had deletion, frameshift mutation, initiation failure or a premature stop codon, rendering them replication-incompetent. In contrast, such mutations were rarely seen in HBV genomes from the two patients with chronic persistent hepatitis after they had developed active or fulminant hepatitis and from the patient with chronic active hepatitis, all of whom had vigorous HBV replication with serum HBV DNA from 10(6) to 10(9)/ml. Unique mutations for amino acid changes were more frequent in HBV genomes with a higher replicative activity. These results indicate two kinds of HBV genomes with an HBeAg-minus phenotype, one with defects seriously affecting viral replication and the other without such defects, which would account for different clinical profiles in carriers with antibody to HBeAg.

Hepatitis B surface antigen (HBsAg) and hepatitis C virus (HCV) RNA were surveyed in patients in Yogyakarta, Indonesia, and their subtypes and genotypes were determined by serological methods and polymerase chain reaction with type-specific primers, respectively. Of 149 patients with chronic liver disease including 24 with chronic hepatitis, 86 with liver cirrhosis, and 39 with primary hepatocellular carcinoma, HBsAg was detected in 40 (27%) and HCV RNA in 48 (32%); one patient was positive both for HBsAg and HCV RNA. Thus, the cause of chronic liver disease was not identified in 62 (42%) patients. Of 58 patients on maintenance hemodialysis, four (7%) were positive for HBsAg and 44 (76%) for HCV RNA. Subtype adw was found in 34 (74%) of 46 HBsAg samples and adr in five (11%); compound subtypes, such as adyw and adyr were detected in the remaining seven (15%). Among HCV RNA samples from 48 patients with chronic liver disease, 23 (48%) were of genotype II, 17 (35%) of genotype III and one (2%) of genotype V, in a distribution strikingly different from that of 44 samples from patients on maintenance hemodialysis, 39 (89%) of which were of genotype I and only one (2%) of genotype II. Genotypes were not classifiable in seven (15%) patients with liver disease and four (9%) patients on hemodialysis despite high HCV RNA titers in them all. These results indicate that different HCV genotypes prevail in patients with distinct diseases, as well as unclassifiable HCV genotypes in Indonesia.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.

A total of 145 patients with chronic liver disease, including 20 with chronic hepatitis, 63 with cirrhosis and 62 with primary hepatocellular carcinoma from Nepal were tested for markers of hepatitis B virus or hepatitis C virus infection. HBsAg was detected in 57 (39%) and hepatitis C virus RNA in 12 (8%); the cause of liver disease was not known in the remaining 76 (52%). HBsAg was found in 5 (1.3%) of 379 normal controls, whereas hepatitis C virus-associated antibodies were detected in 13 (3.4%), none of whom was positive for serum hepatitis C virus RNA. Subtypes of 102 HBsAg samples, from patients and asymptomatic carriers, were adw in 35 (34%), adr in 4 (4%) and ayw in 48 (47%); the remaining 15 (15%) were of atypical subtypes such as ad, ay and a. Of 12 hepatitis C virus RNA samples, genotype I was detected in 1, genotype II in 5 and genotype V in 1; the remaining five samples were not to be classified by polymerase chain reaction with primers specific for genotypes I to V deduced from hepatitis C virus core sequences, despite high hepatitis C virus RNA titers in all of them. Sequences of 192 amino acids in the entire E1 region of unclassifiable hepatitis C virus isolates from five patients differed from each other in 17% to 23%, and varied from reported isolates of defined genotypes in 13% to 44%. These results indicate that atypical subtypes of hepatitis B virus and novel genotypes of hepatitis C virus would prevail in Nepal.

A monoclonal antibody (I-18) was raised against an enneapeptide representing amino acids 125 to 133 of the product of the S gene of hepatitis B virus DNA [S(125-133) segment] with a sequence of Thr-Ile-126-Pro-Ala-Gln-Gly-Thr-Ser-Met. Another monoclonal antibody (T-7) was raised against an S(125-133) segment in which Ile-126 was replaced by Thr-126. In a panel of 16 samples of hepatitis B surface antigen (HBsAg) with known S gene sequences, I-18 reacted with 5 with Ile-126. T-7 reacted with 10 HBsAg samples with Thr-126; it did not, however, react with the remaining one of subtype ayw with Thr-126 flanked by Met-125 and Thr-127. The two allelic subtypic determinants, specified by Ile-126 and Thr-126 and distinct from d/y or w/r, were named i and t after isoleucine and threonine, which regulate them. They were expressed in a mutually exclusive fashion in 216 (83%) of 260 HBsAg samples from asymptomatic carriers. They were not detected in 36 (14%) samples; the failure to detect an i or t determinant was particularly common in HBsAg samples of subtype ayw (26 [79%] of 33). A part of the S gene sequence was determined for eight HBsAg samples without a detectable i or t determinant. They had an Ile-126 or Thr-126 residue that was flanked by Thr-127, not the Pro-127 commonly possessed by HBsAg samples displaying an i or t determinant. Expression of the i/t allele, therefore, would require Pro-127. In eight (3%) of the samples, both i and t determinants were detected; the presence of i and t on the selfsame HBsAg particles was verified by sandwiching the particles between I-18 and T-7. A point mutation from thymine to cytosine at nucleotide 377 in the S gene, contributing different second letters to codon 126 (ATT for Ile and ACT for Thr), would have been responsible for the assembly of HBsAg particles with both i and t determinants by means of phenotypic mixing.

A variant of hepatitis B virus (HBV) having a specific mutation within the S gene has been found to infect vaccinees. To know whether similar variants were involved in Japan, we analyzed two cases of maternal transmission of HBV in infants immunized with hepatitis B immune globulin and hepatitis B vaccine. DNA clones of HBV S genes were propagated from patients and family members and sequenced. In one family, the DNA clones from the baby patient had a Gly-to-Arg mutation at the 145th codon of the S gene, whereas those from her mother had no such mutations. In the other family, all the DNA clones obtained from the two infected children had the 145th codon intact, but they had a missense mutation at the 126th codon of the S gene, causing an amino acid substitution of Asn for Thr or Ile. This same mutation was observed in 12 of 17 clones of DNA obtained from their mother. In comparison with the wild type HBV-derived hepatitis B surface antigen, the two types of S gene mutations, either at the 145th or the 126th codon, were associated with a significant decrease in the antigenicity of some determinants on the hepatitis B surface antigen, measured by MAb. Amino acid substitution at these sites, therefore, would have induced the escape from conventional vaccines that were S gene products of wild type HBV and also from hepatitis B immune globulin, whose main components were probably also antibodies against the S gene products expressed by wild type HBV.

Hepatitis B virus (HBV) DNA was tested for in 294 blood units which had antibody against hepatitis B core antigen (anti-HBc) as the isolated serological marker of HBV infection. After amplification by polymerase chain reaction, HBV DNA was detected in 12 (6.9%) of 175 units that were positive for anti-HBc with hemagglutination inhibition titers greater than or equal to 2(6), significantly more often than in none of 119 units with titers less than or equal to 2(5) (p less than 0.01). These results indicate that the exclusion of blood units with isolated high-titer anti-HBc would be effective for further decreasing the risk of posttransfusion hepatitis B.

Hepatitis B e Ag (HBeAg) was isolated from pooled sera of carriers, without abnormalities in liver function, by affinity column chromatography with mAb against HBeAg. HBeAg polypeptide with an estimated molecular size of 20,000 Da (p20e) was detected, in addition to regular HBeAg polypeptides (p17e/p18e). p20e, as well as p17e/p18e, did not bind with mAb against the carboxyl-terminal domain of the C-gene product. p20e disclosed an N-terminal sequence of MQLFHLXLII- (X unknown), whereas p17e had that of SKLXLGXLXGMDIDPXKEFG- (X's unknown). By comparing them with the amino acid sequence encoded by the precore region and C gene of hepatitis B virus DNA, p20e was deduced to possess amino acids 1 to 19 of the precore-region product at the N-terminus, which contains signal sequence and usually removed before the secretion of HBeAg. p17e had amino acids 20 to 29 of the precore-region product that continued to the C-gene product. Inasmuch as p20e was invariably detected in HBeAg preparations from carriers without evidence for liver disease, it would not have been released into the circulation from destructed hepatocytes. HBeAg polypeptide bearing an uncleaved signal sequence would help in further understanding the mechanism of HBeAg secretion.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.

Clones of hepatitis B virus were propagated from 10 cases of fulminant hepatitis B after amplification by polymerase chain reaction and their nucleotide sequences of the precore region were determined. All 113 clones from 9 cases had a point mutation from guanine to adenine at nucleotide 83 in the precore region, which converted codon 28 for tryptophan (TGG) to a stop codon (TAG) and prohibited the synthesis and secretion of hepatitis B e antigen. Precore-region defects were not detected in any of 23 clones from the remaining 1 case. By contrast, precore-region defects were not found in any of 180 clones from 8 cases of acute hepatitis B without hepatic failure serving as controls. The source of infection was traceable in 3 cases. The same precore-region defect, along with the sequence identity of 435 nucleotides, was observed in clones from the case of a baby and his grandmother, who carried the virus and was implicated in the transmission, and also in clones from two pediatricians and the carrier patients they attended. These findings support the hypothesis that precore-defective mutants have stronger activity to induce fulminant hepatitis than nondefective viruses.

Fulminant hepatitis B developed in 8 recipients of blood units without detectable hepatitis B surface antigen on routine screening. All 124 hepatitis B virus (HBV) DNA clones propagated from their sera possessed defects in the precore region. A point mutation from guanine to adenine at nucleotide 83, converting codon 28 for tryptophan (TGG) to a stop codon (TAG), was the commonest, and it was found in all 113 clones from 7 cases. The remaining case displayed 1 clone with this point mutation and 10 clones with an insertion of 2 base pairs after nucleotide 26. Antibody to hepatitis B core antigen (anti-HBc) was detected in a high titer in 1 of 10 pilot plasma samples of blood units transfused to this case. HBV DNA clones propagated from it exhibited the same precore-region defects as those from the recipient. On the basis of these results HBV mutants, defective in the precore region, would appear to be responsible for posttransfusion fulminant hepatitis B, and the exclusion of blood units with high-titered anti-HBc would be efficacious in preventing it.

Clones of hepatitis B virus (HBV) DNA were propagated from sera of two babies who developed neonatal fulminant hepatitis B, as well as from sera of their mothers who carried HBV with antibody to hepatitis B e antigen, and the precore-region sequences were determined. A point mutation from guanine to adenine, converting codon 28 for tryptophan (TGG) to a stop codon (TAG), was detected in 18 of 20 HBV DNA clones from mother and all 31 clones from baby in one family, and invariably in 55 clones from mother and three clones from baby in the other family. These results indicate that HBV mutants defective in the precore region in some carrier mothers with antibody to hepatitis B e antigen may transmit fulminant hepatitis B to their babies.

Hepatitis B virus DNA clones were propagated from sera of six patients with chronic hepatitis B who seroconverted from HBeAg to antibody to HBeAg either spontaneously or after administration of alpha-interferon. Defects in the precore region blocking synthesis and secretion of HBeAg were detected in all 46 hepatitis B virus DNA clones from three patients who remained positive for antibody to HBeAg and in whom hepatitis resolved. Defective clones had point mutations from guanine to adenine at nucleotide 83 in the precore region, converting codon 28 from tryptophan (TGG) to a stop codon (TAG). In contrast, this defect was not found in any of 39 hepatitis B virus DNA clones from three patients who seroconverted to antibody to HBeAg but then redeveloped HBeAg with reactivation of hepatitis. Using these results, the G-to-A point mutation at nucleotide 83 in the precore region would predict sustained positivity for antibody to HBeAg and remission of hepatitis in patients who have seroconverted either spontaneously or with interferon therapy.

A plasma pool from 12 asymptomatic carriers seropositive for antibody against hepatitis B e antigen (anti-HBe) contained hepatitis B virus (HBV) with chimpanzee infectious doses of 1-100/mL, and another pool from 12 carriers positive for hepatitis B e antigen (HBeAg) contained 10(8)/mL doses or more. The HBeAg-positive pool contained 10(6)-fold more HBV DNA than the anti-HBe-positive pool, reflecting the difference in infectivity in chimpanzees. The precore region sequences of HBV DNA in the two plasma pools were amplified by polymerase chain reaction, and separate HBV DNA clones were propagated for determining the nucleotide sequence. Of 114 clones from the anti-HBe-positive pool, 113 displayed a point mutation from guanine to adenine at nucleotide 83 in the precore region, which converted codon 28 for tryptophan (TGG) to a stop codon (TAG), and the remaining clone had a point mutation from adenine to cytosine at the first letter of codon 1 (CTG) to inhibit the translation initiation of the precore region. Precore region defects, in contrast, were observed in only 10 (8%) of 119 clones from the HBeAg-positive pool. These results indicate the infectious capacity of HBV mutants, defective in the precore region and incapable of directing the synthesis and secretion of HBeAg, which prevail in the circulation of hosts after they seroconvert from HBeAg to anti-HBe.

Some patients with type B chronic active hepatitis have a high titer of hepatitis B virus DNA despite antibody against e antigen in the serum. Clones of hepatitis B virus were propagated from the sera of seven patients with this disease, and the precore region was sequenced. Essentially all clones (128/131 or 98%) showed a point mutation from guanine to adenine at nucleotide 83, converting codon 28 for tryptophan (TGG) to a stop codon (TAG); the second guanine-to-adenine point mutation at nucleotide 86 was identified in only 29 clones from two patients. In patients followed up since they had hepatitis B e antigen, a shift from guanine to adenine was observed at nucleotide 83 along with the seroconversion to the antibody to e antigen. The precore-region product is required for the synthesis and secretion of e antigen from hepatocytes. A point mutation from guanine to adenine at nucleotide 83 observed in the seven patients, therefore, would be responsible for disturbed secretion of e antigen.

The S-gene fragments of hepatitis B virus (HBV) DNA in serum, or integrated in chromosomes of human hepatoma cells (PLC/PRF/5), were amplified by the polymerase chain reaction, cloned into an M13 phage vector, and then sequenced only for adenine. The subtype determinant d or y was established by the presence or absence of adenine as nucleotide 365, and w or r by that of nucleotide 479 in the S gene. The results were identical with those obtained by enzyme immunoassay with monoclonal antibodies. A high sensitivity for the detection of HBV DNA, amplified by the polymerase chain reaction, allowed subtyping of HBV in sera containing HBsAg in concentrations too low to be subtyped by immunological methods. Furthermore, subtyping at the nucleotide level can be applied to tissues containing HBV DNA sequences in integrated forms, such as hepatocellular carcinomas, stored frozen or in formalin.

The C gene of hepatitis B virus (HBV) codes for a nucleocapsid protein made of 183 amino acid residues and is preceded in phase by the precore (pre-C) region, encoding 29 residues. The pre-C-region product is required for the synthesis and secretion of hepatitis B e antigen (HBeAg), which is made of the C-terminal 10 amino acid residues of the pre-C-region product and the N-terminal 149 residues of the C-gene product. HBV mutants with pre-C-region defects prevailed in the circulation of three asymptomatic carriers as they seroconverted from HBeAg to the corresponding antibody (anti-HBe), and these mutants finally replaced nondefective HBV. HBV DNA clones were propagated from sera of an additional 15 carriers with anti-HBe and sequenced for the pre-C region. Essentially all HBV DNA clones (56 of 57 [98%]) revealed mutations that prohibited the translation of a functional pre-C-region product. A point mutation from G to A at nucleotide 83, converting Trp-28 (TGG) to a stop codon (TAG), was by far the commonest and was observed in HBV DNA clones from 16 (89%) of 18 carriers seropositive for anti-HBe. In addition, there were point mutations involving ATG codon to abort the translation initiation of the pre-C region, as well as deletion and insertion to induce frameshifts. Such mutations leading to pre-C-region defects were rarely observed in persistently infected individuals positive for HBeAg or in patients with type B acute hepatitis after they had seroconverted to anti-HBe. These results would indicate a selection of pre-C-defective mutants in persistently infected hosts, along with seroconversion to anti-HBe, by immune elimination of hepatocytes harboring nondefective HBV with the expression of HBeAg.

Sera from 11 patients with fulminant hepatitis B were tested for antibodies to translation products of the pre-S1 and pre-S2 regions of hepatitis B virus of IgM, IgA and IgG classes, as well as of IgA1, IgA2 and SIgA, with solid-phase enzyme immunoassays using native viral polypeptides. Antibodies to pre-S1 region product of IgM and/or IgA class were detected invariably in six patients who still had detectable hepatitis B surface antigen in serum at the time of clinical presentation. The remaining five patients who had lost HBsAg at presentation had antibodies to pre-S region products of various immunoglobulin classes in higher titers. The five patients with fulminant hepatitis without HBsAg had higher levels of IgA antibodies to pre-S region products than the seven patients with nonfulminant acute hepatitis B who had lost HBsAg: IgA antibody to pre-S1 region product (75.6 +/- 63.8 vs. 2.9 +/- 3.2, p less than 0.01) and IgA antibody to pre-S2 region product (28.9 +/- 25.3 vs. 4.2 +/- 6.9, p less than 0.01). IgA antibodies to pre-S1 and pre-S2 region products were invariably polymeric in fulminant hepatitis B. These findings are compatible with the hypothesis that a heightened humoral antibody response to pre-S1 and pre-S2 region products occurs early during the course of fulminant hepatitis B, participating in severe hepatic injury and early clearance of virus characteristic of this disease.

The antibody to hepatitis B core antigen (anti-HBc) is belived to be a marker for natural infection with hepatitis B virus (HBV). In order to study the etiological role of HBV in relation to primary hepatocellular carcinoma (PHC), the anti-HBc in sera of 31 PHC patients was surveyed by the immune adherence hemagglutination method which was about 10 times more sensitive than the complement fixation method. Twenty two out of 31 PHC cases were positive for anti-HBc (71.0%). The is a higher rate of incidence than that of HBs-Ag (51.6%). However, high anti-HBc titer above 2(10) which might reflect current infection with HBV was observed in 15 of these 22 cases. The remaining 7 cases had a titer lower than 2(8); 5 of these patients had neither HBs-Ag nor anti-HBs in their sera. As control, 37 anti-HBs positive blood donors with no definite liver disease were surveyed for anti-HBc titer. Thirty-four of them were positive, but in the majority of cases, the serum titer was less than 2(9), which might only reflect previous infection with HBV. These findings indicate that not only the prevalence of anti-HBc, but also the determination of its real titer is quite important for studying the relationship of HBV to PHC.

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.

The S gene encoding the major surface polypeptide of hepatitis B virus is preceded by the region pre-S(2) with a capacity to code for 55 amino acid residues. In the product of region pre-S(2), the sequence of 19 amino acid residues (amino acids 14-32 from the N terminus) representing an area of high local hydrophilicity is shared by viral strains of subtypes adr, ayw, and ayr; residue 22, phenylalanine, is replaced by leucine in a strain of the other subtype, adw. A synthetic peptide vaccine involving these 19 amino acid residues, when given to two chimpanzees, raised antibodies that bound to viral particles and protected the animals from challenge with 10(6) chimpanzee infectious doses of hepatitis B virus.

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.

The entire nucleotide sequence of genomic DNA was determined for hepatitis B virus (HBV) of subtype ayr, which had been derived from the blood of a Japanese asymptomatic carrier. The genome was 3215 nucleotides long, and differed in DNA sequence by 10% from that of subtypes adw or ayw, but by only 2% from that of subtype adr. Amino acid sequences coded for by the S, C, P and X genes, as well as by the pre-S region, closely resembled those of subtype adr, indicating that the evolution of HBV/ayr from HBV/adr was more recent than the differentiation of the other three subtypes. In the product of the S gene, the mutually exclusive subtypic determinants of the surface antigen, d and y, were associated with variation of amino acid residues at only the 68th and 122nd positions from the N terminus, in contrast to the variation at as many as seven positions for the other set of subtypic determinants, w and r. Sequences representing high local hydrophilicity in the product of the S gene were involved in subtypic variation, although such sequences in the pre-S region were shared by HBV genomes of the various subtypes. In particular, a hydrophilic sequence of 19 amino acid residues, coded for by the pre-S(2) region and implicated in the presumed hepatotropism of HBV, was possessed in common by HBV/adr, HBV/ayr and HBV/ayw, and differed in HBV/adw by only one residue at the 9th position. This amino acid sequence appears to be a promising candidate for a synthetic peptide vaccine.

Small spherical particles produced in the non-permissive phase of hepatitis B virus infection, when the viral genome is integrated into the chromosome of hosts, are rich in the product of the S gene, but poor in the product of the pre-S2 region. For the purpose of adding immunogenicity to spherical particles deficient in the pre-S2 region product, they were conjugated with a synthetic peptide of 19 amino acid residues. The peptide reproduced a hydrophilic area of the pre-S2 region product encoded by viral genomes of subtypes adr, ayw and ayr. The spherical particles supplemented with the pre-S2 peptide raised antibody to the pre-S2 region product in mice, in addition to antibody to the product of the S gene. Antibody to pre-S2 region product, prepared from sera of immunized mice by absorption with the S gene product, bound to spherical particles bearing pre-S2 region product, irrespective of adr, adw, ayw or ayr subtype, and agglutinated hepatitis B virions in immune electron microscopy. Based on the results obtained, the synthetic peptide may prove useful in adding protective efficacy to small spherical particles poor in pre-S2 region product.

A total of 75 clones of hepatitis B virus (HBV) DNA were isolated from the plasma of 4 symptom-free carriers. Each set of clones from a carrier all possessed at least one complete genome, for which the entire nucleotide sequence was determined. A deletion of nucleotide sequence, spanning from 13 to 1071 base pairs, was detected in 17 clones (23%). As many as 16 of them had a deletion in the C gene, with or without the involvement of the P and X genes. The remaining one clone had a deletion restricted to the pre-S region. Accordingly, these defective mutants could not replicate themselves without help from complete HBV co-infecting the same hepatocyte. Defective mutants would have developed during the replication of HBV by reverse transcription of an RNA intermediate, and provide another evidence for a close similarity of HBV with retroviruses.

Hepatitis B subviral particles, purified from plasma of asymptomatic carriers seropositive for hepatitis B e antigen, were treated with various conditions reported for the processing of vaccines. Thereafter, antigenic activities displayed by the translation products of pre-S(1), pre-(2) regions and the S gene were determined with monoclonal antibodies, and the reactivity for polyalbumin receptor was tested. Heating at 100 degrees C for 1.5 min and then at 65 degrees C for 10 h preserved more than 1/2 of antigenic activities representing products of pre-S(1), pre-S(2) regions and the S gene. After incubation in the presence of 8 M urea at 37 degrees C for 4 h, more than 2/3 of antigenic activities still remained. The antigenic activity of the S gene product was decreased to 2/3 and that of pre-S(2) region product to 1/3, after treatment with formalin at the final concentration of 1:4000 at 37 degrees C for 72 h, whereas the activity of pre-S(1) region product was affected drastically. Although 1/5 of the antigenic activity of the S gene product survived the digestion with pepsin for 18 h, antigenic activities of pre-S(1) and pre-S(2) region products were destroyed almost completely. Polyalbumin receptor, borne by the pre-S(2) region product, was lost by pepsin digestion also. Based on the results obtained, heating may be most appropriate for sterilizing plasma-derived hepatitis B particles for use as a vaccine, because it is reliably virucidal and would not affect the protective efficacy to an extent as the other virucidal methods would.

The complete nucleotide sequences of the DNA of three hepatitis B virus (HBV) genomes of subtype adw, cloned from plasma samples of asymptomatic carriers living in the mainland and Okinawa Prefecture of Japan and Indonesia were determined. All three comprised 3215 bp and differed in sequence by only 3.9 to 5.6%. When these isolates were compared with the reported sequences of two HBV genomes of the same subtype derived from American carriers, however, the differences were greater (8.3 to 9.3% to an extent comparable with the nucleotide divergence between an HBV genome of subtype adw and that of a heterotypic subtype, such as adr, ayw or ayr. A total of 18 HBV genomes of various subtypes, including the three described here, 10 reported previously and five unpublished ones, were classified into four groups based on an inter-group divergence in nucleotide sequence of 8% or greater: group A (two adw genomes), group B (four adw), group C (three adw, four adr and one ayr) and group D (four ayw). Thus, the nine genomes of HBV subtype adw were distributed into three groups with considerably different sequences. These results indicate that the four major antigenically defined subtypes of envelope polypeptide do not reflect true genotypic variation of HBV. The fact that d to y, as well as w to r, subtypic change can be induced by an A----G point mutation at nucleotides 365 and 479 in the S gene, respectively, supports this view.

The capsid protein of hepatitis B virus (P19) is made of 183 amino acids and carries the antigenic sites of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) on the amino-terminal domain. The carboxyl-terminal domain of P19 (amino acids 150-183) is arginine-rich (47%) and faces the interior of the nucleocapsid for the binding with DNA. Monoclonal antibody was raised against an antigenic site on this protamine-like region of P19, which was distinct from HBcAg or HBeAg sites, and the novel antigenic site(s) was provisionally designated as hepatitis B inner core antigen (HBicAg). When P19 in a low concn (150 ng/ml) was immobilized on the solid surface, HBicAg sites were preserved, while HBcAg or HBcAg sites were no longer available on it. This allowed the detection of antibodies against HBicAg (anti-HBic), by sandwiching them between immobilized P19 and anti-IgG labeled with horseradish peroxidase. Anti-HBic was detected in sera from HBsAg carriers, typically those seropositive for antibody to HBeAg. A synthetic arginine-rich decapeptide, with a sequence of Arg-Arg-Arg-Gly-Arg-Ser-Pro-Arg-Arg-Arg, representing amino acids 150-159 of P19 and conserved in the majority of reported hepatitis B virus, absorbed the activity to bind with P19 in seven (44%) out of 16 sera containing anti-HBic. These results indicate that the decapeptide carries an HBicAg epitope and the remaining amino acid sequence of the arginine-rich carboxyl terminal domain (160-183) may be responsible for the other HBicAg epitopes.

During a 5-yr period before the introduction of post-exposure immunoprophylaxis of hepatitis B virus (HBV), 363 medical personnel contracted infection after exposure to a contaminated needlestick. Among them, five (1.4%) developed a persistent carrier state, and they were followed for antibodies against translation product of the pre-S2 region (anti-pre-S2) by the enzyme immunoassay with native viral polypeptides. The consecutive seven cases in whom infection was terminated served as controls, In the acute phase, the levels of IgM and IgG anti-pre-S2 were significantly higher in the seven in whom infection resolved than in the five in whom infection persisted (mean sample:normal ratio, 11.1 ± 4.4 vs 2.0 ± 1.3 and 8.7 ± 7.0 vs 3.0 ± 1.1, respectively, p < 0.05). In convalescence, 6 months after the normalization of serum transaminase, IgG anti-pre-S2 was detected in a high tiler in the staff members with resolved infection (27.4 ± 38.5), but was not detectable in those with persistent infection. On the basis of these observations, the intensity of humoral antibody response against the pre-S2 region product is closely correlated with the resolution of infection after exposure to HBV, and anti-pre-S2 persists in the circulation much longer than previously reported. [ABSTRACT FROM AUTHOR]