DNA, Circular ; isolation & purification ; DNA, Viral ; isolation & purification ; Hepatitis B ; diagnosis ; Hepatitis B virus ; genetics ; Humans

China ; epidemiology ; Hepatitis B ; classification ; epidemiology ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Mutation

<b>OBJECTIVEb>To investigate whether the hepatitis B virus (HBV) has quasispecies character by studying nucleotide sequence polymorphism and mutation features of HBV PreC/C gene region, and preliminaryly explore the heterogeneity of HBV quasispecies.

<b>METHODSb>The serum sample was obtained from a patient with chronic hepatitis B, and the whole HBV PreC/C gene region was amplified by PCR and cloned. Thirty-four clones that contained HBV PreC/C gene fragments were sequenced.

<b>RESULTSb>There were 28 kinds of different nucleotide sequences in 34 clones, and the nucleotide sequences diversity ranged from 0.2% to 2.1%. The mutation points were almost distributed in the whole region, but there wasn't mutation at PreC region nt.1 896 point in all sequences.

<b>CONCLUSIONb>Hepatitis B virus has complex quasispecies character in the patients with chronic hepatitis B.

Adult ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Mutation ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

<b>OBJECTIVESb>To study the phenomena of hepatitis B virus (HBV) integration into the tissues of hilar cholangiocarcinoma (HCCA) and to identify the integration sites in the host genome.

<b>METHODSb>Ten fresh HCCA samples were collected from the tissues by surgical ablation, 1 normal hilar bile duct sample selected as control. Cellular DNA were extracted by Wizard SV Genomic DNA Purification System. PCR-derived assay (HBV-Alu-PCR) was employed to amplify the viral-host junctions which contain the HBV sequence and the adjacent cellular flanking sequences. The PCR products were purified and subjected to sequencing by ABI-3730XL Auto DNA Analyzer. The sequence analysis of viral-host junctions was performed by DNASIS MAX 3.0 bioinformatics software. The insertion sites between viral and cellular sequences were identified through homology comparison using NCBI BLAST and MapViewer search.

<b>RESULTSb>In 10 HCCA samples, 5 were demonstrated to have HBV integration fragments with total 6 inserted sites identified. Sequence analysis from viral-host junction showed that HBV X gene inserted into host genome at random distribution with truncated fragments. HBV integration recurrently targeted the unknown region in upstream of CXXC finger protein-1 (CpG-binding protein) gene (4 cases). p53 tumor suppressor gene was also found at the integration site.

<b>CONCLUSIONSb>There is high integration rate of HBV DNA into cellular genome of HCCA. HBV integration is found frequently into or close to cancer-related genes. The findings demonstrate that HBV infection might have association with the pathogenesis of HCCA.

Aged ; Base Sequence ; Bile Duct Neoplasms ; genetics ; virology ; Cholangiocarcinoma ; genetics ; virology ; DNA, Viral ; genetics ; Female ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Virus Integration

Adult ; China ; epidemiology ; Genes, Viral ; Genotype ; Hepatitis B ; epidemiology ; virology ; Hepatitis B virus ; classification ; genetics ; isolation & purification ; Humans ; Male

Genes, Viral ; Genotype ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Polymerase Chain Reaction ; methods

<b>OBJECTIVEb>To identify the intergenotype recombination of hepatitis B virus (HBV) in Chinese patients and explore new recombination types of HBV.

<b>METHODSb>Complete genome sequences of HBV from Chinese patients were downloaded from GenBank and analyzed by Fragment-typing method to screen the potential recombinants, and the breakpoints of recombination were identified by using Simplot program.

<b>RESULTSb>Thirty-one recombination types including 755 recombinants were identified from 1642 complete HBV genome sequences, including 22 B/C recombination types with 676 recombinants, 5 C/D recombination types with 75 recombinants, 3 A/C recombination types with 3 recombinants, and 1 C/I recombination type with 1 recombinant.

<b>CONCLUSIONb>Of the 31 recombination types comprising 755 recombinants, 4 are novel recombination types found for the first time. All these recombination types identified in this study involve genotype C.

Genome, Viral ; Genotype ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Phylogeny ; Recombination, Genetic

<b>OBJECTIVEb>To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).

<b>METHODSb>The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.

<b>RESULTSb>The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.

<b>CONCLUSIONb>The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes ; Genetic Engineering ; Hepatitis Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis E virus ; genetics ; immunology ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Vaccines, Synthetic

<b>BACKGROUNDb>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).

<b>METHODSb>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.

<b>RESULTSb>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.

<b>CONCLUSIONSb>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity

BACKGROUNDS/AIMS: Occult HBV infection is characterized by the presence of HBV infection with undetectable HBsAg. This study was carried out to find out the frequency of HBV infection in HBsAg- negative patients. METHODS: Fifty-six HBsAg-negative patients including 17 anti-HCV positive patients were evaluated. Patients were grouped according to their serological status; group A (anti-HBc+, anti-HBs-, n=16), B (anti-HBc+, anti-HBs+, n=26), and C (anti-HBc-, anti-HBs+/-, n=14). DNA was extracted from frozen liver biopsy specimen, and HBV DNA level was measured with real-time PCR. RESULTS: Overall frequency of detectable intrahepatic HBV DNA was 34% (19/56). The frequency was 56% (9/16) in group A, 31% (8/26) in group B and 14% (2/14) in group C (P=0.01). Intrahepatic HBV DNA levels were as follows; 2,010 +/- 6,660 copies/mg in group A, 6,180 +/- 29,530 copies/mg in group B and 350 +/- 1,220 copies/mg in group C. The frequency of occult HBV infection was not increased in anti-HCV positive patients. CONCLUSIONS: Intrahepatic HBV DNA is frequently detected in anti-HBc positive, HBsAg-negative patients, although the concentration is low.
Middle Aged ; Male ; Liver/*virology ; Humans ; Hepatitis B virus/*genetics/isolation & purification ; Hepatitis B Surface Antigens/*analysis ; Hepatitis B/*diagnosis/immunology/virology ; Female ; DNA, Viral/*analysis ; Aged ; Adult