Sequence variation of hepatitis B virus precore-core open reading frame isolated from serum and liver of children with chronic hepatitis B before and after interferon treatment

Abstract

DNA and amino acid sequences of the hepatitis B virus (HBV) genome were studied in serum and liver samples taken from 12 children with chronic hepatitis B before and after interferon (IFN) therapy. The purpose was to discover whether the persistence of low levels of viral replication with normal alanine aminotransferases after the response to IFN treatment is due to the appearance of mutations in the sequence of HB core antigen T and B cell epitopes. The existence of mutants was studied by amplification of precore-core region of the HBV genome by polymerase chain reaction (PCR) and direct sequencing of the PCR products. In addition to the wild type sequence, mutation 1896 in the precore region was detected in the baseline serum and liver samples of five children. No changes in the distribution were found in the final samples, except one case. In the core region, both the wild type sequence and amino acid substitutions were observed in the basal serum and/or liver samples of six patients and most of these remained detectable in the samples after treatment. Sixteen (67%) of 24 changes in the core amino acid sequences were found in the T- or B-cell epitopes. The results suggest that viral persistence after response to IFN therapy in children is not due to the appearance of mutants in the HBV core T- and B-cell epitopes and that the host immune response can control the viral replication.

Immune and Viral Profile from Tolerance to Hepatitis B Surface Antigen Clearance: a Longitudinal Study of Vertically Hepatitis B Virus-Infected Children on Combined Therapy

“…Nucleotide (synonymous) and amino acid (nonsynonymous) substitutions within universally recognized T helper (Th 1 to 20 and Th 50 to 69), CTL (CTL 18 to 27 and CTL 141 to 151), and B-cell (B 74 to 89, B 107 to 118, and B 130 to 138) immunodominant epitopes were compared with wild-type sequences, and the results were expressed as the number of differences in substitutions (6,7,23,25,33).…”

“…The numbers of all mutations investigated reached a peak at TW28, decreased during the last weeks of combined treatment, and returned to baseline levels during follow-up. No difference in HBV core gene mutation distributions before and after interferon treatment was reported in a small cohort of children with chronic HBV infection and biochemically active disease (6). Similarly, a minimal difference in the number of HBV core gene mutations was found in HBeAg ϩ immunoactive children, who cleared HBeAg spontaneously or after IFN-␣ therapy, compared to children with HBeAg persistence (23).…”

“…Five microliters of DNA template was amplified in the presence of 1ϫ PCR buffer (New England Biotech [NEB], United Kingdom), 2.5 U of Taq polymerase (NEB), 0.25 mM each deoxynucleotide (Sigma Aldrich), and 20 pmol of each primer (Sigma Genosys, United Kingdom) in a final volume of 25 l (6,17,23,25,33). For the HBV core gene region, the external P1 (5Ј-ATA AGA GGA CTC TTG GAC TA-3Ј) and P2 (5Ј-AAA GAC AGG TAC AGT AGA AG-3Ј) primers were used in the firstround reaction on an automated Hybaid NBS 0.2S Satellite Thermocycler (MWG Biotech, Germany).…”