Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy

Since the introduction of antiviral compounds such as lamivudine and famcic lovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV pol ymerase gene has been observed. The selection of these mutations is general ly considered the cause of viral nonresponsiveness and treatment failure, T herefore, the detection of these mutations is of clinical importance. Previ ously genotyped HBV strains isolated from treated and untreated patients we re amplified with primers specific for the I-IBV polymerase region from ami no acids 465 to 562, Amplified products were cloned into plasmid vectors, T he clones were used as reference strains. A set of 38 highly specific oligo nucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected, These probes were applied as 19 different li nes on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon pos itions simultaneously for each clone. PCR products generated from two patie nts infected with HBV genotypes A and C, respectively, and treated with nuc leoside analogs were analyzed on these strips, A gradual increase in geneti c HEY polymerase complexity was observed in follow-up samples compared to t hat in pretreatment samples, Additional analysis of HBV polymerase DNA frag ments in recombinant plasmid clones demonstrated the existence of ii) clone s dth double mutations, iii) clones with single mutations at either codon 5 28, 552, or 555, and (iii) the simultaneous occurrence of two or more,viral populations within one sample, This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of r esistance mutations.