Quantitative DNA fragment analysis for detecting low amounts of hepatitis B virus deletion mutants in highly viremic carriers

Abstract

Many variants of hepatitis B virus (HBV) with deletions in the viral genome have been identified. Some of these variants are indicator or even effector of a more severe course of hepatitis. These deletion mutants contribute a variable and sometimes very low proportion to the viral population. For early detection of small amounts of deletion mutants among a large number of wild-type genomes, we applied a new screening method designated quantitative fragment analysis (QFA). By QFA the whole viral genome can be scanned for the presence of deletions or insertions of ≥3 nucleotides representing more than 2% of the viral population. Using QFA we showed that an often described deletion of 8 nucleotides is packaged in viral capsids and not a polymerase chain reaction (PCR) artifact. QFA was applied to study the emergence of deletion mutants in a group of 18 pediatric patients who had been infected from a common source while being under multidrug cancer chemotherapy. All patients had developed a highly viremic asymptomatic HBV carrier state. In 3 of these patients 3 different kinds of HBV deletion mutants were found by QFA: 8 bp deletions within the core promoter, core gene deletions from 8 to 86 bp, and large deletions of up to 1,989 bp spanning the precore/core and the preS/S reading frames. PCR primers that specifically amplify deletion variants enabled the detection of additional patients harboring the investigated variant.