S. Miska et al., HETEROGENEITY OF HEPATITIS-B VIRUS C-GENE SEQUENCES - IMPLICATIONS FOR AMPLIFICATION AND SEQUENCING, Journal of hepatology, 18(1), 1993, pp. 53-61
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.