Hepatitis B surface antigen particles with all four subtypic determinants: point mutations of hepatitis B virus DNA inducing phenotypic changes or double infection with viruses of different subtypes.

Hepatitis B surface antigen (HBsAg) particles carry the common determinant, a, as well as d or y and w or r subtype determinants, and are classified into the four major subtypes, i.e., adw, adr, ayw and ayr. Rare sera contain HBsAg particles with all four subtype determinants (adywr). Target sequences (nucleotides 38-550) in the S gene of hepatitis B virus (HBV) DNA in two such sera were amplified by the polymerase chain reaction. Individual amplification products were cloned in an M13 phage vector. The HBV DNA clones obtained were subtyped by determining the second letters of codon 122 and 160 for lysine (AAA/AAG) or arginine (AGA/AGG), which specify the d or y and w or r determinants, respectively. From one serum (S-63), two adw, 10 adr and 58 ayr clones were obtained. When the two adw clones and two representatives each of the adr and ayr clones were compared against each other, for the sequence of 235 base pairs representing nucleotides 295-529 in the S gene, they differed only by 0.4-2.1% (average 1.2%). These results indicated multiple point mutations of a single HBV strain of subtype ayr and co-infection of hepatocytes with the original HBV strain and its mutant of subtype adw as the mechanism for the production of HBsAg/adywr particles. From the other serum (K-45), 1 adw, 73 adr and 4 ayw clones were obtained. The adw clone and two representative adr clones differed only by 0-1.7% in the S gene sequences, but they differed by 8.5% or greater from two representative ayw clones. HBsAg/adywr particles in this serum, therefore, could be explained by double infection of hepatocytes with two HBV strains of different subtypes (adr and ayw).