Adult ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Ovary ; virology

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 establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes.

<b>METHODSb>Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group.

<b>RESULTSb>(1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection.

<b>CONCLUSIONSb>Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.

Animals ; DNA Replication ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Gene Expression ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Hepatocytes ; virology ; Male ; Rats ; Rats, Wistar ; Transfection ; Virus Replication

Occult HBV infection (OBI) is defined as presence of HBV DNA in the liver tissue in patients with serologically undetectable HBsAg. There are differences in virologic and serological profiles of OBI. Majority of OBI are positive for anti-HBs and/or anti-HBc and minor portion are negative for all HBV markers. However, there are no HBV mutations in the surface and its regulatory regions. HBV infection persists by the presence of covalently closed circular DNA (cccDNA) within the infected hepatocytes, which serves as a reservoir for future infection. OBI increases the risk of HBV transmission through transfusion, hemodialysis, and organ transplantation. Therefore effective measures should be employed to screen OBI. Antiviral therapy is needed in HBsAg-negative transplant patients who are anti-HBc positive to prevent the recurrence of HBV infection. Since HBV replication is strongly suppressed by immune surveillance system in OBI patients, immunosuppression results in massive HBV replication. This leads to acute hepatitis and sometimes mortality when immune surveillance is recovered after stopping immunosuppressive drugs/anticancer chemotherapy. Therefore, narrow surveillance is required to recognize the viral reactivation and start antiviral agents during immunosuppressive therapy/anticancer chemotherapy in patients with OBI.
Blood Transfusion ; DNA, Viral/analysis ; Hepatitis B/*diagnosis/transmission ; Hepatitis B Core Antigens/immunology ; Hepatitis B virus/genetics/*physiology ; Humans ; Liver Transplantation ; Renal Dialysis ; Virus Activation

<b>OBJECTIVEb>This study aimed to assess the clinical significance of intrahepatic hepatitis B core antigen (HBcAg) (+) in patients with chronic hepatitis B (CHB).

<b>METHODSb>200 CHB patients were prospectively studied using fluorescence quantitative PCR (FQ-PCR), combined PCR with fluorescence probe hybridization technique, to determine serum HBV DNA. Serum HBeAg was measured quantitatively. Liver biopsies were performed and immunohistochemistry stained liver slides were examined in all the cases. Correlation analyses were performed.

<b>RESULTSb>Based on the HBV DNA levels, the patients were divided into 5 groups: group A (<3 log10 copies/ml) n=20, group B (>or=3 log10 copies/ml-<5 log10 copies/ml) n=13, group C (>or=5 log10 copies/ml-<6 log10 copies/ml) n=24, group D (>or=6 log10 copies/ml-<8 log10 copies/ml) n=116, and group E (>or=8 log10 copies/ml) n=27, and 87.5% of the CHB patients were intrahepatic HBcAg (+). The rate of HBcAg (+) was 55.0% (11/20) in group A, 53.8% (7/13) in group B, 75.0% (19/24) in group C, 96.6% (112/116) in group D, and 100% (27/27) in group E. A strong correlation was found between the rate of HBcAg (+) and the level of serum HBV DNA (r=0.80). This type of association also appeared between serum HBV DNA levels and HBeAg (+) (r=0.47). Of 20 CHB patients who were serum HBV DNA negative, 25% (5) were HBeAg (+), and 55% (11) were HBcAg (+), whereas 15 patients were both HBV DNA (-) and HBeAg (-), and 46.7% (7) were HBcAg (+).

<b>CONCLUSIONSb>Intrahepatic HBcAg (+) in CHB patients might be more reliable in reflecting HBV replication. Determination of HBcAg (+) may have clinical significance for evaluating the efficacy of antiviral therapy and for predicting the therapeutic responses to different antiviral agents.

Adult ; DNA, Viral ; blood ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B virus ; immunology ; physiology ; Hepatitis B, Chronic ; immunology ; virology ; Humans ; Liver ; virology ; Male ; Virus Replication ; Young Adult

<b>OBJECTIVEb>To 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.

<b>METHODSb>The 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.

<b>RESULTSb>There 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.

<b>CONCLUSIONb>Hepatitis 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

DNA, Viral ; analysis ; Hepatitis B Core Antigens ; analysis ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Immunohistochemistry ; Liver ; virology ; Liver Cirrhosis ; virology ; Oligonucleotide Array Sequence Analysis ; methods

<b>OBJECTIVEb>To detect the mutation of HBV-DNA open reading frame (ORF), provide evidences for clinical diagnosis and treatment.

<b>METHODSb>By using gene chip technique, HBV-DNA was amplified by PCR, incorporated with fluorescence, hybridized with oligonucleotide to detect the gene sequence of DNA by computer analysis and observe the natural mutation of HBV-DNA ORF.

<b>RESULTSb>The mutations of HBV-DNA ORF existed widespread. The rates of mutation on Pre-C 1896, PreC 1814, BCP 1762, BCP 1764, P 528, P 552MI, P 552MV were 23.5%, 3.9%, 55.9%, 53.9%,39.2%, 38.2%, 10.8%, respectively.

<b>CONCLUSIONb>The gene chip technique possesses extremely high sensitivity and reliability,it is one of the effective methods to detect gene mutation. The mutation of HBV-DNA has important influence on the stability and progress of the disease, and on the judgement of prognosis.

DNA, Viral ; genetics ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; genetics ; immunology ; Humans ; Mutation ; Oligonucleotide Array Sequence Analysis ; methods ; Open Reading Frames ; genetics

BACKGROUND/AIMS: Many patients with positive anti-HBc, but negative HBsAg, are known to harbor occult HBV infection, which may transmit the virus through the graft in liver transplantation. We examined the change of HBV DNA within the liver allograft tissue of the donor with positive anti-HBc, but negative HBsAg, before and after the transplantation and assessed its significance. METHODS: Twenty-eight patients with available posttransplant biopsies that received anti-HBc positive liver allografts between April 2000 and November 2003 were enrolled in the study. Intraoperative wedge biopsy of donor liver and needle biopsy of the recipient around the 12th postoperative day were used. HBV DNA within the liver tissue was identified by polymerase chain reaction technique using paraffin-embedded liver tissue. RESULTS: Among 13 patients that showed positive amplification before transplantation, 10 turned negative and 3 remained positive after transplantation. One patient, who was negative, became positive after transplantation. Three patients had recurrent HBV infection, but none had positive PCR before or after transplantation and recurrence was not associated with PCR results. Donors with low anti-HBs titer were more likely to be PCR positive compared to donors with high anti-HBs serology (P<0.05). CONCLUSIONS: Under adequate prophylactic measures, the presence of HBV DNA within the liver tissue does not affect recurrence and most allografts harboring HBV DNA before transplantation will eventually show viral clearance. However, many anti-HBc positive allografts are infected by HBV at subclinical level so vigilant surveillance is essential.
Middle Aged ; Male ; *Living Donors ; *Liver Transplantation ; Liver/virology ; Humans ; Hepatitis B, Chronic/diagnosis/immunology/virology ; Hepatitis B virus/*genetics ; Hepatitis B Core Antigens/*immunology ; Hepatitis B Antibodies/*analysis ; Female ; DNA, Viral/*analysis ; Adult

<b>OBJECTIVEb>To study the expression of HBsAg and HBcAg in hepatocytes in CHB patients, and analyze the correlation among the expression of HBsAg and HBcAg, the quantity of HBV DNA in serum, the pathology of liver tissue and the clinical manifestation.

<b>METHODSb>Quantitative polymerase chain reaction was used to assay the quantity of HBV DNA in serum in 351 CHB patients. Furthermore pathological diagnosis was performed using liver biopsy to assay the expression of HBsAg and HBcAg in hepatocytes by an immunohistochemical staining technique.

<b>RESULTSb>The positive expression rate of HBsAg and HBcAg in hepatocytes was 92.3% and 76.9% respectively. Cytoplasm-membrane HBcAg expression type (75.6%) was observed in the CHB with more active inflammation, while Nucleus HBcAg expression type (24.4%) was observed in the CHB with more sedative one (P < 0.0001). The expression of HBsAg was correlated with the quantity of HBV DNA in serum (rp = 0.24, P = 0.0129), while inversely correlated with the inflammation and the fibrillation of liver tissue (rp = -0.22, P = 0.0279; rp = -0.23, P = 0.0186). The expression of HBcAg was correlated with the quantity of HBV DNA in serum (rp = 0.52, P < 0.0001), while was inversely correlated with the inflammation and the fibrosis of liver (rp = -0.33, P < 0.0001; rp = -0.34, P < 0.0001).

<b>CONCLUSIONb>Cytoplasm-membrane HBcAg expression type was observed in the CHB with more active inflammation, while Nucleus HBcAg expression type was observed in the CHB with mild change. In the immunopathogenesis of the liver damage in CHB, HBcAg might be a main target antigen. HBsAg might be a sensitive index to screen HBV infection; HBcAg might probably be a reliable index to evaluate the replication of HBV

Adolescent ; Adult ; Child ; Child, Preschool ; DNA, Viral ; blood ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B, Chronic ; immunology ; pathology ; virology ; Hepatocytes ; virology ; Humans ; Immunohistochemistry ; Liver ; pathology ; Male ; Middle Aged