Quantitative DNA fragment analysis for detecting low amounts of hepatitis B virus deletion mutants in highly viremic carriers.
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.
Schläger
F
F
Kinderheilkunde, Justus-Liebig-Universität, Giessen, Germany.
Schaefer
S
S
Metzler
M
M
Gratzki
N
N
Lampert
F
F
Gerlich
W H
WH
Repp
R
R
D000293:Adolescent; D000328:Adult; D000937:Antigen-Antibody Reactions; D016477:Artifacts; D001483:Base Sequence / Q000235:genetics; D002353:Carrier State / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D053938:DNA Fragmentation; D017353:Gene Deletion; D006509:Hepatitis B / Q000276:immunology / Q000635:transmission*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007108:Immune Tolerance; D016133:Polymerase Chain Reaction; D014766:Viremia / Q000235:genetics*; D014771:Virion / Q000235:genetics