AMPLIFICATION OF FULL-LENGTH HEPATITIS-B VIRUS GENOMES FROM SAMPLES FROM PATIENTS WITH LOW-LEVELS OF VIREMIA - FREQUENCY AND FUNCTIONAL CONSEQUENCES OF PCR-INTRODUCED MUTATIONS

To facilitate the investigation of hepatitis B virus (HBV) sequence va riation, we recently established a method for functional analysis of P CR-amplified full-length REV genomes. This study aimed at estimating t he number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-type HBV DNA template mole cules in concentrations like those present in samples from patients wi th very low levels of viremia were amplified, sequenced (30 kb total), and functionally tested. We found that Taq polymerase and a Taq-Pwo p olymerase mixture introduced an average of 5.7 and 3.1 mutations per g enome, respectively, corresponding to polymerase error rates of 12.1 x 10(-5) and 6.0 x 10(-5). One of 8 genomes (12%) amplified with Tag po lymerase, but 7 of 17 genomes amplified with Taq-Pwo polymerases (41%) , remained replication competent. All replication competent genomes ex pressed HBs and HBe antigens and had an average of only 0.9 mutations per genome, In contrast, replication-defective genomes had an average of 5.4 mutations, which frequently also disturbed viral antigen expres sion. From these data we conclude that many of the replication-compete nt HBV genomes from a clinical specimen will retain their replication and antigen expression phenotypes even after extensive amplification w ith Taq-Pwo polymerases. Because replication competence is highly sens itive to random mutations, it is the best marker for the identificatio n of HBV genomes with few or no PCR-introduced mutations.