The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for I-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinapts of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42-and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.

Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide. A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of ZB-galactosidase was expressed and characterized in bacterial extracts. Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X). Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera. The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X. As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection. Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).

A total of 1,915 sera collected in 1979 from asymptomatic hepatitis B surface antigen (HBsAg) carriers were tested for delta antigen, antibody to delta antigen (anti-delta), hepatitis B e antigen (HBeAg) and antibody to hepatitis B e antigen (anti-HBe) in addition to HBsAg and its subtypes. These sera represented blood donated by volunteers to 49 of 57 regions of the American Red Cross located in nine geographic regions of the United States and Puerto Rico. A total of 72 (3.8%) sera had anti-delta activity while none had a detectable level of delta antigen. A significantly higher (p less than 0.01) prevalence of anti-delta (12.1%) was found in San Jose, California (Pacific Region); on the other hand, the East South Central region covering Alabama, Kentucky, Mississippi and Tennessee had a significantly lower (p less than 0.05) prevalence (1.4%) of anti-delta when compared with all other regions combined. Anti-delta was, however, detected in all regions of the United States and in Puerto Rico. The cause of significant differences in the prevalence of anti-delta was not clear. The distribution of anti-delta was not associated with age, sex or blood type of the donor. Sixty-nine of 70 samples with anti-delta were found among the 1,527 samples that had either HBeAg or anti-HBe. And among 149 that lacked both HBeAg and anti-HBe, only one sample had anti-delta. The difference is statistically significant (p less than 0.05). The presence of anti-delta was not associated with HBsAg/ad (2.7%) or HBsAg/ay (4.6%).

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