A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.
Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.
Günther
S
S
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Hamburg, Federal Republic of Germany.
Li
B C
BC
Miska
S
S
Krüger
D H
DH
Meisel
H
H
Will
H
H
D001483:Base Sequence; D017931:DNA Primers; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D017353:Gene Deletion; D016679:Genome, Viral; D006509:Hepatitis B / Q000276:immunology*; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007165:Immunosuppression Therapy; D016030:Kidney Transplantation / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014771:Virion / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D017931:DNA Primers; D004251:DNA Transposable Elements; D004279:DNA, Viral