Occult HBV infection is characterized by the absence of serum HBsAg with persistence of low level of intrahepatic HBV DNA. Several suggested mechanisms for the origin of occult HBV infection include strong suppression of viral replication and gene expression, mutation in the regulatory regions of HBV genome, formation of immunoglobulin-bound HBsAg, viral interference, and blockage of HBsAg secretion from infected hepatocytes. Standardized assays are not yet available, and sensitive HBV DNA amplification assay is necessary for the diagnosis of cryptic infection. Detection rate of HBV DNA is highest in IgG anti-HBc positive population. However, neither anti-HBc nor anti-HBs can be detected in a significant proportion of infected persons. Occult HBV infection occurs in a number of clinical settings and is highly prevalent in HCV-infected patients as well as in patients with cryptogenic chronic liver disease including hepatocellular carcinoma.
DNA, Viral/analysis ; Hepatitis B/*diagnosis/*epidemiology/metabolism ; Hepatitis B Antibodies/blood ; Hepatitis B Core Antigens/immunology ; Hepatitis B Surface Antigens/blood ; Humans

<b>OBJECTIVEb>To investigate the effects of the hepatitis B virus (HBV) P22e protein on the apoptosis of human hepatocellular carcinoma HepG2 cells.

<b>METHODSb>HepG2 cells were transfected with recombinant plasmid pEGFP-HBVP22e and exposed to Act-D and tumor necrosis factor alpha (TNFalpha) treatment to induce cell apoptosis. Flow cytometry was performed to determine the proportion of cells containing sub-G1 DNA to represent the number of apoptotic cells. Laser scanning confocal microscopy was used to observe the nuclear alterations in the apoptotic cells.

<b>RESULTSb>HepG2EGFP-C2HBVP22e cell strain showed a much delayed apoptosis as well as obviously lowered apoptotic rate in comparison with the HepG2 strain (P<0.01).

<b>CONCLUSIONb>The introduction and expression of extraneous gene HBVP22e significantly inhibits the apoptosis of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Humans ; Transfection ; Viral Core Proteins ; metabolism

Adult ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B, Chronic ; drug therapy ; immunology ; virology ; Humans ; Liver ; metabolism ; Male ; Middle Aged ; Vidarabine ; therapeutic use

<b>OBJECTIVEb>To develop a coexpression plasmid which expressing envelope protein and nucleoprotein of hepatitis B virus and know of its expressing efficiency.

<b>METHODSb>The plasmid coexpressing envelope protein and nucleoprotein of hepatitis B virus under the CMV promoter respectively was constructed by gene recombination. Cellular expression was assessed by ELISA.

<b>RESULTSb>Multiple cloning site was inserted into expression vector contain hepatitis B virus PreS2-S gene. And expression unit containing hepatitis B virus PreC-C was cloned into it. HBsAg and HBeAg was detected both in the culture supernatant and in the cells.

<b>CONCLUSIONb>The coexpressing plasmid was constructed successfully and it can express effectively in vitro. This has provided a basis for further research of the therapeutic HBV DNA vaccine.

Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; Humans

<b>OBJECTIVEb>To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.

<b>METHODSb>Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution.

<b>RESULTSb>HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15,000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein.

<b>CONCLUSIONb>HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.

Furin ; metabolism ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Humans ; Microdissection ; Microscopy, Confocal ; Proprotein Convertases ; metabolism ; Transfection ; Virus Replication

<b>OBJECTIVEb>To test whether nuclear factor kappa B plays an important role in the apoptosis-inhibitory effect of hepatitis B virus (HBV) P22(e) protein.

<b>METHODSb>HepG2 cells were transfected with recombination plasmid pEGFP-HBVP22(e). The Act-D and TNF alpha were used to induce apoptosis. NF-kappa B inhibitor ALLN were used to inhibit the signaling pathway. The activation of NF-kappa B was EMSA, and the nulear translocation of NF-kappa B was determined by immuno-staining.

<b>RESULTSb>Laser scanning confocal microscopy and EMSA indicated that HBV P22(e) protein enhanced the nuclear translocation of NF-kappa B after apoptosis induction. ALLN treatment inhibited the nuclear translocation of NF-kappa B, and blocked the apoptosis-inhibiting effect of HBV P22(e) protein.

<b>CONCLUSIONb>This study indicates that HBV P22(e) protein inhibits apoptosis of hepatocyte via the NF-kappa B signaling pathway.

Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; Leupeptins ; pharmacology ; Liver Neoplasms ; metabolism ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Plasmids ; Signal Transduction ; drug effects ; Transfection ; Viral Core Proteins ; metabolism

<b>OBJECTIVEb>To investigate whether insertion of TC motif into hepatitis B virus (HBV) core protein c/e1 site affects the expression of S and e antigen.

<b>METHODSb>Different oligonucleotides encoding TC motif were inserted into the c/e1 site of the core gene of a 1.3 copy wild-type HBV genome vector. HepG2 cells were divided into several groups of cells to transiently transfect with the wild-type and mutant HBV vectors, respectively. In each group, the expression level of core protein inside cells was detected by western blotting, and the levels of S and e antigen in culture medium were analyzed by ELISA assay.

<b>RESULTSb>Western blotting showed that these TC-tagged core proteins were expressed at similar level of wild-type one. ELISA assay indicated that the level of S and e antigen in culture medium of different groups were not significantly different.

<b>CONCLUSIONb>Insertion of TC motif into HBV core protein c/e1 site does not interference with the expression of viral protein encoded by HBV genome.

Amino Acid Sequence ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; Mutagenesis, Insertional ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Subcellular localizaton of HBcAg have been found to be related to the activity of liver disease and HBV replication. The aim of this study was to determine whether the degree of expression of HBcAg in the hepatocyte nucleus and cytoplasm reflects the level of viral replication and histological activity in chronic HBV infection. A total of 102 patients with biopsy proven chronic hepatitis B were included. There was a highly significant correlation between the levels of HBV DNA in serum and the degree of expression of HBcAg in the nucleus for HBeAg-positive(p=0.000) and negative patients(p=0.04). There was a highly significant, correlation between the degrees of expression of HBcAg in hepatocyte cytoplasm and histologic activities (p<0.01) for HBeAg-positive patients. The degrees of expression of HBcAg in the hepatocyte cytoplasm correlated positively with the lobular activities (p<0.01), but not correlated with the portal activity and fibrosis for HBeAg-negative patients. In conclusion, in the young patients with chronic B viral hepatitis, the degree of expression of HBcAg in the hepatocyte nucleus may affect viral load, and the degree of expression of HBcAg in the hepatocyte cytoplasm may affect histologic activities of liver disease.
Male ; Liver/pathology/virology ; Humans ; Hepatocytes/pathology/*virology ; Hepatitis B, Chronic/*pathology/*virology ; Hepatitis B e Antigens/metabolism ; Hepatitis B Core Antigens/*metabolism ; DNA, Viral/blood ; Cytoplasm/virology ; Cell Nucleus/virology ; Adult ; Adolescent

<b>OBJECTIVEb>To study the influence of HBV/P22 protein on the induced apoptosis of HepG2 cells.

<b>METHODSb>In vitro, two HepG2 strains were transfected with pcDNA3.1+ and pcDNA3.1+HBV/P22 respectively and the cells were exposed to Act D and TNF alpha for 6h and then the induced apoptosis was detected by flow cytometry (FCM) and TUNEL technique. Supernatant HBeAg was detected by Abbott reagent. The intracellular expression of HBV/P22 protein was measured by Western blot and immunochemistry. In vivo, three cell groups were inoculated into nude mice by subcutaneous injections. After two weeks, Act D and TNF alpha were injected into the tumors and then the induced apoptosis in the tissues was detected by TUNEL technique. The expression of HBV/P22 protein in the tumor tissues was detected by immunochemistry.

<b>RESULTSb>In vitro, in HepG2- pcDNA3.1+HBV/P22 cells, supernatant HBeAg was positive and intracellular HBV/P22 protein was positively expressed. The apoptosis proportion of HepG2-pCDNA3.1+HBV/P22 cells was markedly lower than HepG2 and HepG2-pcDNA3.1+ cells (P < 0.05). In vivo, HBV/P22 protein was expressed in the tumor tissues, and the apoptosis proportion in the group injected with HepG2-pcDNA3.1+HBV/P22 cells was markedly lower than those injected with HepG2 and HepG2-pcDNA3.1+cells (P < 0.05).

<b>CONCLUSIONb>HBV/P22 protein could inhibit the induced apoptosis of HepG2 cells both in vitro and in vivo.

Animals ; Apoptosis ; Female ; Hep G2 Cells ; Hepatitis B Core Antigens ; genetics ; Hepatitis B e Antigens ; metabolism ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transfection ; Viral Core Proteins ; genetics

<b>OBJECTIVESb>The hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle.

<b>METHODSb>Dimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method.

<b>RESULTSb>The HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative.

<b>CONCLUSIONb>In HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.

Chromosome Positioning ; Dimethyl Sulfoxide ; pharmacology ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Neoplasm Metastasis ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; Viral Core Proteins ; metabolism ; Virus Assembly