Gene Products, tat ; genetics ; Gene Targeting ; Hepatitis B virus ; genetics ; growth & development ; Humans ; Recombinant Fusion Proteins ; genetics ; Ribonucleases ; genetics ; Transduction, Genetic ; Virus Replication ; drug effects

BACKGROUNDS/AIMS: Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. METHODS: Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. RESULTS: Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). CONCLUSIONS: Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.
Cell Line, Tumor ; Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/*genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/*antagonists & inhibitors/genetics/metabolism ; *Virus Replication

No abstract available.
Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/antagonists & inhibitors/genetics ; *Virus Replication

<b>OBJECTIVEb>To establish a culture system of HBV positive serum infected Hep G2 cells in vitro.

<b>METHODSb>Hep G2 cells were seeded into six-well cluster dishes, at 1 x 10(-6) cells per well and incubated with 3 ml 10% fetal calf serum/ Dulbecco's modified Eagle's medium (10% FCS/DMEM) at 37 degrees in 5% CO2 air. At 24 h after plating, infection group Hep G2 cells were cultured with 0.5 ml HBV positive serum, in control group HBV negative serum was used, 24 h later the inoculums was removed. The cells were then extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After washing with PBS, 4 ml 2% FCS/DMEM were added to each well and the medium was collected every 12 h. ELISA method was used to detect HBsAg in culture medium. HBV DNA in cells and culture medium was detected by PCR.

<b>RESULTSb>In infection group, HBsAg could be detected from cell culture medium from 12 h (after PBS washed) to 84 h. HBV DNA could be detected by PCR in culture medium and cells.

<b>CONCLUSIONb>Infection of Hep G2 cells by HBV positive serum is feasible.

Carcinoma, Hepatocellular ; pathology ; virology ; Cell Line, Tumor ; DNA, Viral ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; blood ; virology ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; genetics ; growth & development ; immunology ; Humans ; Liver Neoplasms ; pathology ; virology ; Polymerase Chain Reaction ; Serum ; virology

<b>OBJECTIVEb>To detect the level of serum and liver tissue TGF-beta1 in patients with chronic hepatitis B, to study their relation to liver fibrosis and gain the evidence for diagnosis of liver fibrosis.

<b>METHODSb>The liver fibrosis grades (S0-S4) of 131 cases with chronic HBV infection were diagnosed after liver biopsy. Serum TGF-beta1 was detected by enzyme-linked immunosorbent assay, and the semiquantitative analysis was applied after detecting the expression of TGF-beta1 in liver tissue with immunohistochemistry. Their relations to liver fibrosis were analyzed.

<b>RESULTSb>Serum and tissue level of TGF-beta1 increased significantly with the development of fibrosis, and the same result was obtained between themselves (P < 0.01). There was very significant difference for serum level of TGF-beta1 among the groups with different fibrosis grades (P < 0.01). Serum levels of TGF-beta1 were decreased significantly comparing the Group S0 or S1 to S4 (P < 0.005). There were significant difference for serum level of TGF-beta1 among S0 and the others (P < 0.005). And there was significant difference between S1 and S3 (P < 0.005). The expression level of TGF-beta1 in liver tissue has no significant difference between group S3 and S4 (P > 0.05). However, the differences were significantly among the other comparisons (P < 0.01).

<b>CONCLUSIONb>There is close relation between the level of TGF-beta1 and the different liver fibrosis grades due to chronic hepatitis B. The serum level of TGF-beta1 is a potential noninvasive maker for diagnosis of liver fibrosis.

Adult ; Female ; Hepatitis B ; complications ; drug therapy ; metabolism ; pathology ; Hepatitis B virus ; genetics ; metabolism ; Hepatitis B, Chronic ; complications ; metabolism ; Humans ; Liver Cirrhosis ; etiology ; Male ; Transforming Growth Factor beta1 ; blood ; metabolism

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

<b>OBJECTIVEb>To investigate the alteration of HBV markers in liver allograft of HBV related recipients pre and post liver transplantation under Lamivudine or combination of Lamivudine with HBIG prophylaxis and explore the mechanism of HBV de nova infection in liver allograft after orthotopic liver transplantation, as well as seek to establish a optimal prophylactic protocol.

<b>METHODSb>The serial liver biopsy specimens of 90 liver allograft and sera of 78 liver transplant recipients during operation and after 1 week, 1 month, 3 months, 6 months, 12 months, 24 months post transplantation have been collected and detected for HBV markers with enzyme-linked radioimmunoassay, fluorescent quantitative assay for HBV-DNA in serology and with immunohistochemistry stain, HBV-DNA in situ hybridization in histology for detection of HBV markers in liver allograft samples.

<b>RESULTSb>Whether recipients with active replicative or inactive replicative HBV preoperatively, none of positive HBV-DNA, HBsAg and HBcAg in 100% liver biopsy specimens with HBV-DNA hybridization in situ and immunohistochemistry stains in histology within 2 hours after reperfusion.

<b>CONCLUSIONb>Whatever HBV replicative status the recipients have before surgery, no evidence of HBV particles direct invasion to the liver allograft from HBV related cirrhotics during operation under current prophylactic measures. However, the further supposed mechanism and its significance in HBV de nova infection of liver allograft remained to be disclosed further.

Adolescent ; Adult ; Aged ; Biomarkers ; blood ; Child ; DNA, Viral ; blood ; Female ; Hepatitis B Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; growth & development ; Hepatitis B, Chronic ; diagnosis ; drug therapy ; surgery ; Humans ; Immunization, Passive ; Immunoglobulins ; administration & dosage ; Lamivudine ; therapeutic use ; Liver Transplantation ; Male ; Middle Aged ; Preoperative Care ; methods ; Secondary Prevention

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; virology ; Genes, p53 ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; virology ; Loss of Heterozygosity ; Nuclear Proteins ; metabolism ; Point Mutation ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism

Transforming growth factor (TGF)-beta1 is a key cytokine producing extracellular matrix. We evaluated the effect of TGF-beta1 gene polymorphism at codon 10 on the development of cirrhosis in patients with chronic hepatitis B. One hundred seventy eight patients with chronic hepatitis (CH, n=57) or liver cirrhosis (LC, n=121), who had HBsAg and were over 50 yr old, were enrolled. The genotypes were determined by single strand conformation polymorphism. There were no significant differences in age and sex ratio between CH and LC groups. HBeAg positivity and detection rate of HBV DNA were higher in LC than in CH groups (P=0.055 and P=0.003, respectively). There were three types of TGF-beta1 gene polymorphism at codon 10: proline homozygous (P/P), proline/leucine heterozygous (P/L), and leucine homozygous (L/L) genotype. In CH group, the proportions of P/P, P/L, and L/L genotype were 32%, 51%, and 17%, respectively. In LC group, the proportions of those genotypes were 20%, 47%, and 33%, respectively. The L/L genotype was presented more frequently in LC than in CH groups (P=0.017). Multivariate logistic regression analysis confirms that detectable HBV DNA (odds ratio [OR]: 3.037, 95% confidence interval [CI]: 1.504-6.133, P=0.002) and L/L genotype (OR: 3.408, 95% CI: 1.279-9.085, P=0.014) are risk factors for cirrhosis.
Aged ; Asian Continental Ancestry Group/genetics ; *Carrier State ; *Codon ; Female ; Genetic Predisposition to Disease ; Genotype ; Hepatitis B virus/genetics ; *Hepatitis B, Chronic/genetics/virology ; Humans ; *Liver Cirrhosis/genetics/virology ; Male ; Middle Aged ; Odds Ratio ; *Polymorphism, Genetic ; Risk Factors ; Transforming Growth Factor beta1/*genetics

<b>BACKGROUNDb>MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18 - 25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown.

<b>METHODSb>miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A.

<b>RESULTSb>Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A.

<b>CONCLUSIONb>HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.

Blotting, Northern ; Cell Line, Tumor ; metabolism ; virology ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; HLA-A Antigens ; metabolism ; Hepatitis B virus ; growth & development ; physiology ; Humans ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis