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

OBJECTIVETo explore the variations of gene C in hepatitis B viruses between hepatitis B patients and healthy carriers, and provide experimental evidences for analysis of virus gene mutations acting on the virus material science and response of the body to the virus.

METHODSThe virus DNA load in hepatitis B patients and healthy blood donors was investigated by real-time polymerase chain reaction (PCR). Gene sequence analysis was taken to detect gene polymorphism, and all the success samples were compaired with standard strain by DNAstar.

RESULTS(1)G Compared with standard strain, C region in all samples had mutations, there were 31 mutations in at least 2 samples (3 mutations in gene PreC and 28 mutations in gene C), including 9 missense mutations, 1 chain termination mutation and 21 synonymous mutation. Mutations nt 1827 c-->a and nt 2221 c-->t existed in all the samples, and most samples had 6 synonymous mutations. Four hepatitis B patients had mutation nt1896 g-->a, and another 4 patients had 2 mutations, namely, S87G and I97F (or 197L) in HBcAg CTL recognition episome. (2) The success ratio of amplification and sequencing of HBV DNA was closely associated with its copy numbers. In the present study, copy numbers of HBV DNA which were successfully amplified and sequenced were almost more than 40 193/ml.

CONCLUSIONSHBV genome were easily affected by nucleotide mutations, 2 residues had mutations in gene of C region, which is firstly reported, suggesting these mutations may be geographical restricted. Mutations in gene of C region may either change the structure and function of HBeAg and HBcAg, which may further induce the escape of immune clearance for HBV or influence the detection of HBsAg or HBeAg, which may creat new problems for the prevention, diagnosis and treatment of hepatitis B.

Female ; Hepatitis B ; virology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Mutation ; Polymorphism, Genetic

OBJECTIVETo establish a novel improved loop-mediated isothermal amplification (LAMP) technique to detect hepatitis A virus (HAV).

METHODSA novel improved LAMP assay based on the addition of an acceleration primer was developed for hepatitis A virus nucleotide acid detection.

RESULTSPrecision and reproducibility analysis proved its high stability and reliability. Comparison between the improved and conventional LAMP assays revealed that the former was more sensitive with a detection limit of 5 TCID50/ml. The novel detection method displayed 100% consistency with the TaqMan real-time PCR assay when applied to clinical specimens collected from patients with confirmed acute HAV infection.

CONCLUSIONThis novel technique is widely applicable as a simple diagnostic tool in the clinical field as well as for the surveillance and investigation of the infectious disease in developing countries where HAV is endemic.

DNA Primers ; genetics ; Hepatitis A ; virology ; Hepatitis A virus ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; methods ; RNA, Viral ; genetics ; metabolism

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

OBJECTIVESTo 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.

METHODSTen 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.

RESULTSIn 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.

CONCLUSIONSThere 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

OBJECTIVETo analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China.

METHODSThe nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region.

RESULTSThe nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains.

CONCLUSIONAll the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.

Capsid Proteins ; genetics ; China ; epidemiology ; Hepatitis A ; epidemiology ; virology ; Hepatitis A virus ; classification ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo 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).

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe 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

OBJECTIVETo identify the intergenotype recombination of hepatitis B virus (HBV) in Chinese patients and explore new recombination types of HBV.

METHODSComplete 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.

RESULTSThirty-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.

CONCLUSIONOf 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

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThere 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.

CONCLUSIONHepatitis 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

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