Quantitative analysis of wild-type and HBeAg minus hepatitis B viruses by a sequence-dependent primer extension assay.
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.
Brunetto
M R
MR
Laboratory of the Department of Gastroenterology, Molinette Hospital, Torino, Italy.
Randone
A
A
Ranki
M
M
Jalanko
A
A
Piantino
P
P
Giarin
M
M
Capra
G
G
Calvo
P L
PL
Oliveri
F
F
Bonino
F
F
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
D017931:DNA Primers; D004279:DNA, Viral; D006513:Hepatitis B e Antigens