QUANTITATIVE-ANALYSIS OF WILD-TYPE AND HBEAG MINUS HEPATITIS-B VIRUSES BY A SEQUENCE-DEPENDENT PRIMER EXTENSION ASSAY

The ratio between wild-type hepatitis B virus (HBV) and HBV mutant, un able to secrete ''e'' antigen (HBeAg minus HBV) appears to be an impor tant determinant of the outcome of chronic hepatitis B. Quantitative a nalysis of wild-type and HBeAg minus HBVs in the blood could be useful to monitor chronic hepatitis B patients. We developed a solid-phase m inisequencing assay for both viruses using a primer-guided incorporati on of a single labeled nucleotide on an affinity captured biotinylated amplified HBV-DNA template. A standard curve was constructed by mixin g increasing quantities of wild type and mutant virus DNAs. The detect ion of wild-type and HBeAg minus sequences, ranging from 10% to 90% of overall viremia, was linear and reproducible till 0.1 pg/mu l of seru m HBV-DNA. The assay yields numerical values and the ratio of incorpor ated nucleotides defines the relative proportions (%) of the two viral sequences with accuracy. We tested the sensitivity and accuracy of th e minisequencing on mixed end point dilutions of wild-type and HBeAg m inus reference sera and amplified products. The feasibility and reprod ucibility of the assay were tested in 35 sera from 21 HBsAg positive p atients with chronic hepatitis B using both minisequencing and oligo-h ybridization assays. A high correlation was found between the two assa ys (r = 0.957 P < 0.0001). In conclusion, the minisequencing assay pro vides a precise and reproducible quantitative analysis of wild-type an d HBeAg minus HBVs in clinical specimens. It is proposed to study the relations between HBV heterogeneity and the course of hepatitis B and its response to therapy. (C) 1994 Wiley-Liss, Inc.