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 explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.

<b>METHODSb>The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.

<b>RESULTSb>DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.

<b>CONTROLb>0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.

<b>CONTROLb>0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.

<b>CONCLUSIONb>Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

Carrier Proteins ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Down-Regulation ; Gene Expression Regulation ; Hep G2 Cells ; Hepatitis B Surface Antigens ; isolation & purification ; Hepatitis B e Antigens ; isolation & purification ; Hepatitis B virus ; physiology ; Humans ; Interleukin-6 ; metabolism ; NF-kappa B ; metabolism ; Plasmids ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Virus Replication

In recent years, Hepatitis B virus (HBV) persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is concerned. We studied and compared the efficacy among HBV persistent infection mice models by other serotypes except AAV8. First, we prepared and purified five viruses: rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV and rAAV9-1.3HBV. Then we injected each virus into 3 C57BL/6J mice with the dose of lx 1011 vg (Viral genome, vg) per mouse. We detected HBsAg and HBeAg in sera by enzyme-linked immunosorbent assay (ELISA) at different time points post injection. We killed mice 8 weeks post injection and took blood and livers for assay. We detected copies of HBV DNA by real-time quantitative PCR in sera and livers. Meantime, we detected HBcAg in the livers of mice by immunohistochemistry and further performed pathology analysis of these livers. The five groups of mice, HBeAg and HBsAg expression sustained 8 weeks in serological detection and HBV DNA was both detected in sera and livers at the time of 8 weeks post injection. HBeAg, HBsAg, HBV DNA copies expression levels in descending order were AAV8>AAV9>AAV1>AAV5>AAV2. HBcAg expression was detected in livers as well. Varied degrees of liver damage were shown in five groups of mice. This study provides more alternative AAV vector species to establish a persistent infection with hepatitis B model.
Animals ; Dependovirus ; classification ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Mice ; Mice, Inbred C57BL ; Serogroup ; Virus Replication

MicroRNA polymorphisms may be associated with carcinogenesis or immunopathogenesis of infection. We evaluated whether the mircoRNA-604 (miR-604) polymorphism can affect the persistence of hepatitis B virus (HBV) infection, and the development to hepatocellular carcinoma (HCC) in patients with chronic HBV infection. A total of 1,439 subjects, who have either past or present HBV infection, were enrolled and divided into four groups (spontaneous recovery, chronic HBV carrier without cirrhosis, liver cirrhosis and HCC). We genotyped the precursor miR-604 genome region polymorphism. The CC genotype of miR-604 rs2368392 was most frequently observed and T allele frequency was 0.326 in all study subjects. The HBV persistence after infection was higher in those subjects with miR-604 T allele (P=0.05 in a co-dominant and dominant model), which implied that the patients with miR-604 T allele may have a higher risk for HBV chronicity. In contrast, there was a higher rate of the miR-604 T allele in the chronic carrier without HCC patients, compared to those of the HCC patients (P=0.03 in a co-dominant model, P=0.02 in a recessive model). The T allele at miR-604 rs2368392 may be a risk allele for the chronicity of HBV infection, but may be a protective allele for the progression to HCC in chronic HBV carriers.
Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Carcinoma, Hepatocellular/etiology/*genetics/pathology ; Case-Control Studies ; Demography ; Female ; Gene Frequency ; *Genetic Predisposition to Disease ; Genotype ; Hepatitis B Antibodies/blood ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/metabolism ; Hepatitis B, Chronic/complications/*genetics/virology ; Humans ; Liver Neoplasms/etiology/*genetics/pathology ; Male ; MicroRNAs/*genetics/metabolism ; Middle Aged ; Polymorphism, Single Nucleotide ; Risk Factors

Vertical transmission from mother to child, the main route of chronic hepatitis B virus (HBV) infection in the East Asia, is considered one of the most important predictors for the response to antiviral therapies as well as its complications such as cirrhosis and hepatocellular carcinoma. Therefore, it is critical in both etiologic and prognostic aspects to confirm whether or not chronic HBV infection is acquired vertically. This study investigated whether mother-to-child infection could be proved by the phylogenetic analyses of HBV pre-S/S genes ever since several decades have elapsed in mother-child pairs with presumed vertical transmission. The pre-S and S regions of HBVs were compared and analyzed phylogenetically in a total of 36 adults (18 mother-child pairs) with chronic HBV infection. All of the isolates of HBV were genotype C and serotype adr. The divergence between mothers and offsprings was 0 to 1.5%. Phylogenetic trees revealed that 17 of 18 pairs (94%) with presumed vertical transmission were grouped into the same cluster. Vertical transmission from mother to child could be strongly suggested even in adults with a history of several decades of HBV infection using the phylogenetic analyses of pre-S and S genes.
Adult ; Aged ; DNA, Viral/analysis ; Female ; Genotype ; Hepatitis B Surface Antigens/classification/*genetics ; Hepatitis B virus/classification/*genetics/metabolism ; Hepatitis B, Chronic/diagnosis/*virology ; Humans ; Infectious Disease Transmission, Vertical ; Male ; Middle Aged ; Mothers ; Phylogeny ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Serotyping ; Young Adult

<b>OBJECTIVEb>To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

<b>METHODSb>The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.

<b>RESULTSb>The TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.

<b>CONCLUSIONb>A Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication

BACKGROUND/AIMS: Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. METHODS: Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. RESULTS: Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. CONCLUSIONS: Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect.
Adult ; Aged ; Amino Acid Sequence ; Antiviral Agents/therapeutic use ; DNA, Circular/*analysis ; Female ; Genotype ; Hep G2 Cells ; Hepatitis B/drug therapy/*pathology/virology ; Hepatitis B Surface Antigens/blood/*genetics/metabolism ; Hepatitis B virus/genetics/*metabolism ; Humans ; Liver/virology ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Remission, Spontaneous ; Republic of Korea ; Serotyping

No abstract available.
DNA, Circular/*analysis ; Female ; Hepatitis B/*pathology ; Hepatitis B Surface Antigens/*genetics ; Hepatitis B virus/*metabolism ; Humans ; Male

BACKGROUND/AIMS: Carnitine and vitamin complex (Godex(R)) is widely used in patients with chronic liver disease who show elevated liver enzyme in South Korea. The purpose of this study is to identify the efficacy and safety of carnitine from entecavir combination therapy in Alanine aminotransferase (ALT) elevated Chronic Hepatitis B (CHB) patients. METHODS: 130 treatment-naive patients with CHB were enrolled from 13 sites. The patients were randomly selected to the entecavir and the complex of entecavir and carnitine. The primary endpoint of the study is ALT normalization level after 12 months. RESULTS: Among the 130 patients, 119 patients completed the study treatment. The ALT normalization at 3 months was 58.9% for the monotherapy and 95.2% for the combination therapy (P<0.0001). ALT normalization rate at 12 months was 85.7% for the monotherapy and 100% for the combination group (P=0.0019). The rate of less than HBV DNA 300 copies/mL at 12 months was not statistically significant (P=0.5318) 75.9% for the monotherapy, 70.7% for the combination and it was. Quantification of HBsAg level was not different from the monotherapy to combination at 12 months. Changes of ELISPOT value to evaluate the INF-gamma secretion by HBsAg showed the increasing trend of combination therapy compare to mono-treatment. CONCLUSIONS: ALT normalization rate was higher in carnitine complex combination group than entecavir group in CHB. Combination group was faster than entecavir mono-treatment group on ALT normalization rate. HBV DNA normalization rate and the serum HBV-DNA level were not changed by carnitine complex treatment.
Adult ; Alanine Transaminase/blood ; Antiviral Agents/*therapeutic use ; Carnitine/*therapeutic use ; DNA, Viral/analysis ; Drug Therapy, Combination ; Enzyme-Linked Immunospot Assay ; Female ; Guanine/*analogs & derivatives/therapeutic use ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/*drug therapy ; Humans ; Interferon-gamma/metabolism ; Male ; Middle Aged ; Mitochondria/physiology ; Treatment Outcome ; Vitamin B Complex/*therapeutic use

<b>OBJECTIVEb>To investigate the influence of hepatitis B virus (HBV)-encoded small surface protein (SHBs) on hepatic cell expression of host genes related to lipid metabolism.

<b>METHODSb>The full-length SHBs gene was amplified from HBV genotype C by polymerase chain reaction (PCR) and cloned into the pcDNA3.1(+) expression vector for stable transfection into HepG2 cells (selected by G418 screening); cells transfected with empty vector served as control. The SHBs mRNA and protein levels were detected by reverse transcription-PCR and enzyme-linked immunosorbent assay. SHBs effects on expression of genes and proteins related to lipid metabolism were detected by real-time quantitative (q)PCR and western blotting, respectively.

<b>RESULTSb>The stably transfected cell line HepG2-pn3.1-SHBs was established successfully. qPCR showed that the HepG2-pn3.1-SHBs cells had significantly down-regulated transcription of the ECHS1, APOA1 and LPL genes (0.161+/-0.043 vs. control cells: 0.210+/-0.022, t = 2.479; 0.031+/-0.007 vs. 0.094+/-0.055, t = 2.752; 0.770+/-0.036 vs. 0.982+/-0.031, t = 10.914), but significantly up-regulated ACC and SREBP-1c genes (0.113+/-0.027 vs. 0.059+/-0.022, t = -3.757; 0.019+/-0.002 vs. 0.015+/-0.001, t = -4.330). The CPT1a and PPARa genes' expression was slightly, but not significantly, down-regulated in the HepG2-pn3.1-SHBs cells (0.028+/-0.005 vs. 0.030+/-0.004, t = 1.022; 0.014+/-0.004 vs. 0.015+/-0.002, t = 0.758). Western blotting showed similar expression trends for the corresponding proteins.

<b>CONCLUSIONb>SHBs alters the expression of some host genes with known functions in fatty acid synthesis and decomposition; however, it remains unclear whether the hepatitis B surface antigen can directly contribute to development of hepatic steatosis.

Gene Expression ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Lipid Metabolism ; genetics ; Polymerase Chain Reaction ; Transfection