title: Quantitative analysis of wild-type and HBeAg minus hepatitis B viruses by a sequence-dependent primer extension assay.
abstract: The ratio between wild-type hepatitis B virus (HBV) and HBV mutant, unable to secrete "e" antigen (HBeAg minus HBV) appears to be an important determinant of the outcome of chronic hepatitis B. Quantitative analysis of wild-type and HBeAg minus HBVs in the blood could be useful to monitor chronic hepatitis B patients. We developed a solid-phase minisequencing assay for both viruses using a primer-guided incorporation of a single labeled nucleotide on an affinity captured biotinylated amplified HBV-DNA template. A standard curve was constructed by mixing increasing quantities of wild type and mutant virus DNAs. The detection of wild-type and HBeAg minus sequences, ranging from 10% to 90% of overall viremia, was linear and reproducible till 0.1 pg/microliter of serum HBV-DNA. The assay yields numerical values and the ratio of incorporated nucleotides defines the relative proportions (%) of the two viral sequences with accuracy. We tested the sensitivity and accuracy of the minisequencing on mixed end point dilutions of wild-type and HBeAg minus reference sera and amplified products. The feasibility and reproducibility of the assay were tested in 35 sera from 21 HBsAg positive patients with chronic hepatitis B using both minisequencing and oligo-hybridization assays. A high correlation was found between the two assays (r = 0.957 P < 0.0001). In conclusion, the minisequencing assay provides a precise and reproducible quantitative analysis of wild-type and HBeAg minus HBVs in clinical specimens. It is proposed to study the relations between HBV heterogeneity and the course of hepatitis B and its response to therapy.
authors.affiliation: Laboratory of the Department of Gastroenterology, Molinette Hospital, Torino, Italy.
authors.forename: M R
authors.initials: MR
authors.lastname: Brunetto
authors.forename: A
authors.initials: A
authors.lastname: Randone
authors.forename: M
authors.initials: M
authors.lastname: Ranki
authors.forename: A
authors.initials: A
authors.lastname: Jalanko
authors.forename: P
authors.initials: P
authors.lastname: Piantino
authors.forename: M
authors.initials: M
authors.lastname: Giarin
authors.forename: G
authors.initials: G
authors.lastname: Capra
authors.forename: P L
authors.initials: PL
authors.lastname: Calvo
authors.forename: F
authors.initials: F
authors.lastname: Oliveri
authors.forename: F
authors.initials: F
authors.lastname: Bonino
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005821:Genetic Techniques; D006509:Hepatitis B / Q000821:virology; D006513:Hepatitis B e Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D006521:Hepatitis, Chronic / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014766:Viremia / Q000821:virology
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D006513:Hepatitis B e Antigens