Sensitive method for identification of minor hepatitis B mutant viruses.
There is increasing evidence for selection of hepatitis B virus (HBV) mutants during the natural course of infection, after immunization, and during interferon therapy. These mutants can prolong viral persistence, escape immune-mediated elimination, and influence the clinical sequelae of infection. Here we describe a sensitive and reliable method that allows detection of HBV mutants within a heterogeneous virus population. The method consists of amplification of HBV DNA with vector-HBV hybrid primers, efficient and enzyme-free cloning of the amplified DNA fragment, identification by hybridization with synthetic oligonucleotide probes, and sequencing of the cloned DNA fragments. We demonstrate that up to 10(5) HBV DNA fragments can be obtained in cloned form from a single amplification reaction, the ratio of HBV pre-C "wild-type" and mutant sequences of heterogeneous populations can be determined, and very minor virus subpopulations can be identified. The method can be applied to other viral or nonviral nucleic acid sequence variation studies and should help to resolve the role of mutant and wild-type viruses in pathogenesis and the forces driving their selection.
Grötzinger
T
T
Max-Planck-Institut für Biochemie, Martinsried/Munich, Germany.
Will
H
H
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D004279:DNA, Viral