Detection of hepatitis B virus by using polymerase chain reaction and nonradioactive DNA probes. II. Identification of mutations in the core gene by PCR-direct sequencing and ASO probe method

Abstract

Mutants of hepatitis B virus (HBV) in the pre-core/core gene exist in patients chronically infected with HBV. The core gene of HBV DNA is preceded by the pre-core region which takes a role of synthesis and secretion of HBe antigen (HBeAg). Previous studies have suggested that products of the core gene could be immunological targets of cytotoxic T lymphocytes as well as those of variant pre-core genes and subsequently, that the clinical courses of HBV carriers might be influenced due to infected HBV variants. In this study, we therefore examined mutations in the core gene of HBV DNA on strains with or without a translational stop codon at the 28th codon of the pre-core region, using allele-specific oligonucleotide (ASO) probes and PCR-direct sequencing. The analysis of nucleotide sequences (codon 27-100) of HBV DNA in anti-HBe positive sera showed that there were two hypervariable segments of codon 31-49 and codon 87-97, where amino acid substitutions of L31I, S49T, S87G/N, K96N and I87F/1 frequently occurred regardless of the presence or absence of the mutation in the pre-core region. Meanwhile, less mutations were detected in the core gene of HBV DNA in HBeAg positive sera. Furthermore, we could identify the mutations at codon 87, 96 and 97 by using nonradioactive probes in a good coincidence with results by PCR-direct sequencing. The ASO probe method is useful for detection of mutations in the core gene in many specimens.