Amino Acid Sequence ; Hepatitis B Surface Antigens ; classification ; genetics ; immunology ; Hepatitis B virus ; immunology ; Mutation

<b>OBJECTIVEb>To determine the main genotype of hepatitis B virus (HBV) detected from Tibetans in China and provide basic data for hepatitis control and prevention.

<b>METHODSb>The S gene and C gene were amplified by PCR from the sera of HBsAg positive Tibetans. After sequencing, the gene sequences were analyzed and the phylogenetic trees were drawn by the software MEGA3.

<b>RESULTSb>In trees based on S gene, the sequences of most samples clustered at genotype D, while in trees based on C gene, the sequences of all samples clustered at genotype C.

<b>CONCLUSIONb>The dominant genotype of HBV detected from Tibetans in China is a C/D hybrid.

Genotype ; Hepatitis B ; blood ; epidemiology ; virology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; genetics ; Hepatitis B virus ; classification ; genetics ; immunology ; Humans ; Phylogeny ; Tibet ; epidemiology

<b>OBJECTIVEb>To investigate whether the presence of HBV mutant in vaccinees simply reflects the prevalence of HBV mutant in a specific geographic area or is indeed due to the immune pressure induced by vaccination.

<b>METHODSb>HBV S genes were amplified by using polymerase chain reaction (PCR) and DNA sequence analysis of the "a" determinant was performed on sera from 30 childhood patients with immunoprophylaxis and 30 patients without vaccinations.

<b>RESULTSb>Mutations of the "a" determinant were detected in 8 of the 60 patients. They were all of the adw subtype. The prevalence of amino acid substitutions as detected by direct sequencing was higher in those fully-vaccinated than of those not vaccinated. In all 8 vaccinated and also with detectable mutants, the mean age was older than the other vaccinated children.

<b>CONCLUSIONb>The prevalence of mutants is related to HBV subtypes and genotypes. Universal vaccination has accelerated an accumulation of HBsAg "a" determinant mutants with amino acid changes critical for immune escape in vaccinated children who became carriers. This suggests that new vaccination strategies should be considered.

Child ; China ; Epitopes ; genetics ; Female ; Hepatitis B ; prevention & control ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; genetics ; Humans ; Male ; Point Mutation ; Vaccination

<b>OBJECTIVEb>To know genotypes and serotypes of hepatitis B virus (HBV) detected from hepatitis B infected people in Yunnan Province.

<b>METHODSb>Serum samples were collected from HBsAg carriers detected from people who had a physical examination at Yunnan Provincial Center for Disease Control and Prevention. The S genes of HBV were amplified by nested PCR and the PCR products were sequenced. The viral genotype was identified by phylogenetic analysis. 27 reference sequences corresponding to HBV genotype A to I were obtained from GenBank. According to the amino acid sequences deduced from the nucleotide sequences of S gene, the dominant serotype of HBV detected from these people were confirmed.

<b>RESULTSb>39 HBsAg positive serum samples were detected from 2216 people who had a physical examination. The results shows that 76.9% were C genotype; 15.4% were B genotype; 5.1% were D genotype; 2.5% were I genotype. Three serotypes were found. The rates of adw2, adrq+ and ayr serotypes are 71.8%, 17.9% and 10.3% respectively. All of adw2 subtype specimens are C genotype. Among the serum specimens in which both HBsAg and HBeAg are positive, 75% were C genotype and adw2 subtype.

<b>CONCLUSIONb>It is determined that the main genotype and subtype of HBV prevailed in Yunnan province is C genotype and adw2 subtype.

Antibodies, Viral ; immunology ; China ; Female ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Male ; Physical Examination ; Population Surveillance

Animals ; Hepatitis B Surface Antigens ; blood ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B, Chronic ; virology ; Humans ; Protein Precursors ; blood ; genetics ; immunology

<b>OBJECTIVESb>To screen proteins in hepatocytes interacting with hepatitis B virus surface antigen middle protein (MHBs) with yeast-two hybrid technique for studying the biological functions of MHBs.

<b>METHODSb>Polymerase chain reaction (PCR) was used to amplify the gene of MHBs from the plasmid A7 containing the whole fragment of adr subtype of HBV and the PCR product was cloned into pGEM-T vector and then evaluated by sequencing. The gene of MHBs was cut by EcoRI and BamH I from pGEM-T vector and then cloned into the yeast expression plasmid pGBKT7. MHBs bait plasmid was constructed by ligating MHBs gene with yeast expression vector pGBKT7 with yeast-two hybrid system 3 and then was transformed into yeast AH109 (a type). The transformed yeast cells were mated with yeast Y187 (alpha type) containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X- alpha -gal for selecting and screening. After extracting and sequencing the plasmid from true positive blue colonies, the results were analyzed by bioinformatics.

<b>RESULTSb>A pGBKT7- MHBs yeast expressed vector was successfully constructed. Two colonies were sequenced. One colony was Homo sapiens aldolase B fructose-bisphosphate, the other was a new gene with unknown function, which was named MHBs-binding protein 1.

<b>CONCLUSIONb>MHBs gene was successfully cloned. Two genes of MHBs interacting proteins in hepatocytes were obtained by yeast-two hybrid system 3. Our results brought some new clues for studying the biological functions of MHBs and the mechanisms of HBV carcinogenesis.

Genome, Viral ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; Humans ; Two-Hybrid System Techniques

<b>OBJECTIVEb>To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.

<b>METHODSb>Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody.

<b>RESULTb>The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA.

<b>CONCLUSIONb>The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.

Animals ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; analysis ; genetics ; immunology ; Hepatitis B Core Antigens ; analysis ; genetics ; immunology ; Hepatitis B Surface Antigens ; analysis ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; ultrastructure ; Protein Engineering ; Rabbits ; Virion ; chemistry ; genetics ; immunology ; ultrastructure

<b>OBJECTIVEb>This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection.

<b>METHODSb>Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive.

<b>RESULTSb>DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.

<b>CONCLUSIONSb>Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

Base Sequence ; Blood Donors ; DNA, Viral ; analysis ; Genotype ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Antibodies ; Hepatitis B Core Antigens ; isolation & purification ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Protein Precursors ; genetics

<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

Alanine Transaminase ; blood ; DNA, Viral ; blood ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; virology ; Humans