HETEROGENEITY OF HEPATITIS-B VIRUS C-GENE SEQUENCES - IMPLICATIONS FOR AMPLIFICATION AND SEQUENCING

Occasionally direct sequencing of amplified hepatitis B virus DNA lead s to weak signals on autoradiograms. Using amplified C-gene sequences we investigated whether this is due to sequence heterogeneity of virus populations and use of inappropriate primers for direct sequencing. H igh C-gene sequence heterogeneity (point mutations, stop codons and a one codon deletion) was observed in HBV genomes from serum of a chroni c carrier who underwent interferon treatment. The type of C-gene mutat ions detected by direct sequencing depended on the type of primers use d. Cloning and sequencing of amplified C-gene sequences demonstrated t hat this was due to mutations in the region complementary to the seque ncing primer. These data demonstrate the existence of novel HBV C-gene mutants and imply that multiple or degenerate sequencing and amplific ation primers are essential for accurate evaluation of the extent of H BV C-gene heterogeneity. Based on comparative sequence analysis of all available completely or incompletely sequenced C-genes, guidelines fo r optimal primer design are proposed for similar studies.