A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients

Abstract

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.

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

“…A previous study has demonstrated that linear monomeric HBV genomes with SapI ends can initiate a full replication cycle, leading to viral antigen expression. 20 Our results showed that pSuper/HBVS1 could also suppress expression of HBsAg and HBeAg of HBV genotypes B and C from clinical isolates to a similar extent as on a laboratory clone of genotype A ( Figure 5).…”

“…19 To clone the full-length HBV genome from clinical samples, a method described by Gunther et al was used. 20 After amplication, PCR products were purified and cloned into the vector yT&A (Yeastern Biotech Co., Shijr, Taiwan).…”

“…Purification of HBV DNA from extracellular viral particles was performed following the protocol described in the previous study. 20 One third of the DNA purified was fractionated in a 1% agarose gel, blotted to a nylon membrane, and hybridized with a DIG-labeled DNA fragment covering the entire surface gene.…”

In vitro activity of cepharanthine hydrochloride against clinical wild-type and lamivudine-resistant hepatitis B virus isolates

“…HBV DNA was extracted from 200 μl of serum samples using a commercial viral DNA extraction kit (OMEGA, USA). The full-length HBV genome was amplified by polymerase chain reaction (PCR) using the primers and thermocycling conditions described by Günther et al (1995). The PCR product was digested with SapI (MBI, USA) and then ligated into the SapI-digested and shrimp alkaline phosphatase-treated pHY106 plasmid, producing a recombinant clone termed pHY106-HBV.…”

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma

“…To amplify the full sequence of the HBV genome, two pairs of synthetic oligonucleotide primers (p1, p2 and p3, p4) were used. PCR was carried out according to the methods described previously, (12) with LA Taq (TaKaRa Bio Inc., Ohtsu, Japan) for 40 cycles in the first PCR and 35 cycles in the second PCR. The sequences between the joint points of the amplified PCR fragments were amplified using the precore/core primers (p5, p6 and p7, p8).…”