Subtyping hepatitis B virus DNA in free or integrated forms by amplification of the S-gene sequences by the polymerase chain reaction and single-track sequencing for adenine.

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.