Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.