Latent hepatitis B virus infection in healthy individuals with antibodies to hepatitis B core antigen

Several recent reports have shown that hepatitis B virus (HBV) could be frequently transmitted to the recipients from donors who have antibodies to hepatitis B core antigen (anti-HBc) through liver transplantation. We provide here the molecular evidence of latent HBV infection accompanied with ongoing viral replication in the liver tissue of anti-HBc-positive healthy individuals. HBV DNA was detectable in 13 of 14 healthy donors who were positive for both anti-HBc and antibodies to hepatitis B surface antigen (anti-HBs), but in none of 3 who were positive for anti-HBs alone. The detected HBV genomes from these subjects included covalently closed circular DNA and pregenomic RNA, the replication intermediate of HBV. Notably, 5 of 7 cases tested were predominantly infected with wild type HBV strains without any mutations in the precore and core promoter regions under the presence of circulating antibody to hepatitis B e antigen. Interestingly, a predominant clone detected in one donor showed a 63-nucleotide deletion in the precore region including an encapsidation signal sequence. Our findings indicate that the majority of healthy individuals positive for anti-HBc, which had been assumed to denote a past history of transient HBV infection, were latently infected with the episomal form of HBV accompanied by ongoing viral replication and few nucleotide mutations in the precore and core regions.     

载脂蛋白质B mRNA编辑酶催化多肽3G抑制乙型肝炎病毒和鸭乙型肝炎病毒复制

目的了解载脂蛋白质BmRNA编辑酶催化多肽3G（APOBEC3G）对乙型肝炎病毒（HBV）和鸭乙型肝炎炎病毒（DHBV）复制的抑制作用。方法从健康人外周血单个核细胞提取RNA，逆转录聚合酶链反应扩增APOBEC3G，将产物克隆到pXF3H载体的EcoRⅠ和Hind Ⅲ酶切位点以构建真核表达质粒；以ayw亚型HBV全长质粒构建具有复制能力的1．3倍HBV质粒（pHBV1．3）。不同剂量的APOBEC3G真核表达质粒与pHBV1．3共转染HepG2细胞；酶联免疫吸附法检测细胞培养上清液的乙型肝炎表面抗原和e抗原水平，Southernblot和Northernblot分析HBV核衣壳相关DNA和RNA的水平变化。不同剂量APOBEC3G真核表达质粒与头尾相接的2倍DHBV质粒共转染LMH鸡肝癌细胞，Southernblot分析DHBV核衣壳相关DNA水平变化。结果成功构建APOBEC3G真核表达质粒和具有复制能力的1．3倍HBV质粒。APOBEC3G抑制乙型肝炎表面抗原和e抗原的分泌，转染细胞内HBV核衣壳相关RNA表达水平下降，而对核心蛋白质的表达没有影响；APOBEC3G对转染细胞内HBV和DHBV核衣壳相关DNA水平具有剂量依赖的抑制效应。结论APOBEC3G对HBV和DHBV复制具有抑制作用。     

Significance of the expression of pre-S proteins in mononuclear blood cells in chronic hepatitis caused by the hepatitis B virus

In chronic viral type B hepatitis, the presence in the serum of pre-S proteins of hepatitis B virus (HBV) envelope reflects viral replication. As peripheral blood mononuclear cells (PBMC) are known to be target cells for HBV replication, the aim of our study was to investigate the clinical relevance of pre-S protein expression in PBMC. Fifty-seven patients with chronic type B hepatitis and HBs antigenemia were studied. Following separation using the Ficoll gradient, the PBMC were lysed and studied for pre-S proteins by Western blot. HBs Ag and HBc/e Ag were assayed in parallel by radioimmunoassay. HBs Ag was detected in PBMC in 86 percent of cases, HBc/e Ag in 28 percent of cases and pre-S proteins in 34 percent of cases. A statistically significant association was found between the presence of HBc/e Ag in PBMC and both the serum HBe Ag (chi 2 test, p less than 0.01) and the serum viral DNA/DNA polymerase (t test, p = 2.10(-4)). The pre-S protein expression in PBMC was significantly associated with higher levels of DNA/DNA polymerase activity (chi 2 test, p less than 0.05). The expression of pre-S proteins in PBMC appears therefore to correlate with the HBV viral replication phase. The HBc/e Ag and pre-S protein detection in PBMC therefore offers a reliable non invasive approach to tissular viral replication. The clinical relevance of pre-S testing in PBMC was illustrated by the study of 12 cases of chronic active hepatitis positive for anti-HBe but with no or low level of serum DNA polymerase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication

AIM: To investigate whether DNA-dependent activator of interferon-regulatory factors (DAI) inhibits hepatitis B virus (HBV) replication and what the mechanism is.METHODS: After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plasmid, viral protein (HBV surface antigen and HBV e antigen) secretion was detected by enzyme-linked immunosorbent assay, and HBV RNA was analyzed by real-time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 (IRF3) phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-κB (NF-κB) activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Transwell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines.RESULTS: Viral protein secretion was significantly reduced by 57% (P < 0.05), and the level of total HBV RNA was reduced by 67% (P < 0.05). The viral core particle-associated DNA was also dramatically down-regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-κB signaling was essential for DAI to elicit antiviral response in Huh7 cells. When the NF-κB signaling pathway was blocked by a NF-κB signaling suppressor (IκBα-SR), the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was independent of IRF3 signaling and secreted cytokines.CONCLUSION: This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is associated with activation of NF-κB but independent of IRF3 and secreted cytokines.     

Isolated hepatitis B core antibody is associated with HIV and ongoing but not resolved hepatitis C virus infection in a cohort of US women

To characterize predictors of isolated hepatitis B core antibody (anti-HBc) among human immunodeficiency virus (HIV)-infected and HIV-uninfected women, we compared 702 women with anti-HBc and hepatitis B surface antibody (anti-HBs) with 490 women with isolated anti-HBc (1.8% of whom had detectable hepatitis B virus [HBV] DNA). Factors independently associated with isolated anti-HBc without viremia were detectable hepatitis C virus (HCV) RNA, HIV positivity, history of injection drug use, >10 lifetime sex partners, and HIV RNA level >100,000 copies/mL. Anti-HBs levels were lower among anti-HCV-positive women. Isolated anti-HBc was rarely explained by occult HBV in this cohort but may be explained by the influence of viral coinfections on anti-HBs level or durability.     

Serologic markers of hepatitis B virus (HBV) and hepatitis D virus infection in carriers of hepatitis B surface antigen who are frequently exposed to HBV

Serum hepatitis B e antigen (HBeAg) and HBV DNA are indicators of active replication of HBV, whereas IgM antibody to hepatitis B core antigen (IgM anti-HBc) may indicate an active immune response to chronic HBV infection. Fifty-eight carriers of hepatitis B surface antigen (HBsAg) who had frequent parenteral exposures were studied for the presence of HBeAg, HBV DNA, IgM anti-HBc and hepatitis delta virus (HDV) serologic markers. Active replication of HBV was detected in 36.2% (25% of drug addicts, 16.7% of thalassemia patients, and 46.9% of hemodialysis patients) and seropositivity for IgM anti-HBc in 55.2% of the HBsAg carriers. Among the 39 HBsAg carriers who were negative for HBeAg, IgM anti-HBc was detected significantly more frequently than HBV DNA (46.1% vs. 5.1%, p less than 0.001). Serologic evidence of HDV infection was detected in 35% of drug addicts, 50% of thalassemia patients and in 9.4% of hemodialysis patients. These data revealed that continued replication of HBV was more frequent in hemodialysis patients than in drug addicts and thalassemia patients who are HBsAg carriers and the opposite was true for the prevalence of HDV infection.     

乙型肝炎病毒裸DNA转染原代大鼠肝细胞模型的建立

目的建立乙型肝炎病毒(HBV)环化裸DNA转染原代大鼠肝细胞瞬时表达模型,为进一步研究肝细胞与HBV之间的互动关系奠定基础. 方法采用原代大鼠肝细胞(PRH)作为靶细胞,电转环化HBV DNA,于转染后1～10d各时点分别以Southern杂交和斑点杂交分析HBV DNA的复制中间体与复制形式,以IMX系统检测HBsAg、HBeAg,以western免疫印迹、免疫斑点印迹和免疫细胞化学法检测HBcAg,用RT-PCR检测HBV S/X mRNA,并采用电镜观察有无Dane颗粒合成.以单纯电击PRH为对照组. 结果 HBV环化裸DNA在PRH中的复制为游离型,可见rcDNA、cccDNA和ssDNA等复制中间体;表达的蛋白质产物、HBsAg于转染后各时点PRH裂解液中均可检测到(P/N值4.83～85.69,阳性≥2.1),峰值于1～3d,转染后1～10d平均P/N值为18.239±27.459;PRH培养上清液中未检测到HBsAg;HBeAg于PRH培养上清液和细胞裂解液中均呈阴性(P/N值＜2.1);HBcAg于转染后1～3d时点内检测到低度表达;HBV S mRNA为阳性,而X mRNA为阴性;电镜未观察到Dane颗粒. 结论 HBV环化裸DNA转染原代大鼠肝细胞瞬时表达模型稳定性、可重复性均良好.     

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs). METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012 copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group. RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group. CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.     

Pathogenesis of occult chronic hepatitis B virus infection

Occult hepatitis B infection(OBI) is characterized by hepatitis B virus(HBV) DNA in serum in the absence of hepatitis B surface antigen(HBsAg) presenting HBsAg-negative and anti-HBc positive serological patterns.Occult HBV status is associated in some cases with mutant viruses undetectable by HBsAg assays;but more frequently it is due to a strong suppression of viral replication and gene expression.OBI is an entity with world-wide diffusion.The failure to detect HBsAg,despite the persistence of the viral DN...     

Determination of hepatitis B virus DNA incidence, viral load, and mutations in blood donors with HBsAg and anti-HBs-negative serology and antibodies to hepatitis B core antigen

BACKGROUND: The aim of the present study was to determine hepatitis B virus DNA incidence, viral load, and mutations in blood donors with HBsAG and anti-HBs negative serology and antibodies to hepatitis B core antigen. METHODS: Blood samples were collected from 1000 blood donors with HBsAg-negative test results. Anti-HBc total screening was performed using the ELISA method. HBsAg-negative/anti-HBc total-positive samples were tested for anti-HBs, anti-HBc IgM, HBeAg, and anti-HBe. Samples with isolated anti-HBc were determined for viral load of HBV DNA using real-time PCR. RESULTS: Anti-HBc total was established as positive in 200 (20%) of the 1000 blood donors. While anti-HBs was negative in 59 (29.5%) of the 200 anti-HBc-positive samples, it was found to be positive in 141 (70.5%) of them. All of the other hepatitis B markers were negative in all of the anti-HBs-negative samples. HBV DNA was not detected in the sera of the isolated anti-HBc-positive blood donors with real-time PCR. CONCLUSION: Although we could not detect HBV DNA in the sera of the isolated anti-HBc-positive blood donors, findings in the literature suggest that anti-HBc testing should be used in combination with HBsAg for the screening of blood donors to reduce risk. After that, the most appropriate algorithm to follow can be HBV DNA screening of donors.     

Hepatitis B virus reactivation in hepatitis B virus surface antigen negative patients receiving immunosuppression: A hidden threat

AIM: To present the characteristics and the course of a series of anti- hepatitis B virus core antibody (HBc) antibody positive patients, who experienced hepatitis B virus (HBV) reactivation after immunosuppression. METHODS: We retrospectively evaluated in our tertiary centers the medical records of hepatitis B virus surface antigen (HBsAg) negative patients who suffered from HBV reactivation after chemotherapy or immunosuppression during a 3-year period (2009-2011). Accordingly, the clinical, laboratory and virological characteristics of 10 anti-HBc (+) anti-HBs (-)/HBsAg (-) and 4 anti-HBc (+)/antiHBs (+)/HBsAg (-) patients, who developed HBV reactivation after the initiation of chemotherapy or immunosuppressive treatment were analyzed. Quantitative determination of HBV DNA during reactivation was performed in all cases by a quantitative real time polymerase chain reaction kit (COBAS Taqman HBV Test; cut-off of detection: 6 IU/mL). RESULTS: Twelve out of 14 patients were males; median age 74.5 years. In 71.4% of them the primary diagnosis was hematologic malignancy; 78.6% had received rituximab (R) as part of the immunosuppressive regimen. The median time from last chemotherapy schedule till HBV reactivation for 10 out of 11 patients who received R was 3 (range 2-17) mo. Three patients (21.4%) deteriorated, manifesting ascites and hepatic encephalopathy and 2 (14.3%) of them died due to liver failure. CONCLUSION: HBsAg-negative anti-HBc antibody positive patients can develop HBV reactivation even 2 years after stopping immunosuppression, whereas prompt antiviral treatment on diagnosis of reactivation can be lifesaving.     

Hepatitis B virus DNA in hepatitis B surface antigen-positive blood donors: relation to the hepatitis B e system and outcome in recipients

The presence of hepatitis B virus (HBV) DNA was investigated in 26 hepatitis B surface antigen-positive blood donors. Three donors (12%) were concordantly positive for HBV DNA and hepatitis B e antigen (HBeAg) and had IgM antibody to hepatitis B core antigen (anti-HBc). Two donors (8%) had HBV DNA without HBeAg; both were positive for antibody to HBeAg and lacked IgM anti-HBc. Twenty-one HBV DNA-negative donors had antibody to HBeAg, and all were negative for HBeAg and IgM anti-HBc. Blood units from 16 donors were transfused. A sufficient serological and clinical follow-up was available for 10 HBV-susceptible recipients. Three recipients of HBV DNA-positive blood units were infected irrespective of HBeAg status or presence of IgM anti-HBc. Six (86%) of seven recipients of HBV DNA-negative blood units developed HBV infection. Thus all hepatitis B surface antigen-positive blood donors should still be considered infectious irrespective of status with regard to HBeAg, HBV DNA, and IgM anti-HBc.     

Enhanced duck hepatitis B virus gene expression following aflatoxin B1 exposure

Epidemiological studies have suggested synergistic interactions between chronic hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure in the etiology of hepatocellular carcinoma (HCC), although the molecular mechanisms of their interactions are still not understood. The aim of this study was to use the Pekin duck model to investigate the impact of AFB1 exposure on duck hepatitis B virus (DHBV) replication during the early stages of virus-carcinogen interactions. Six-week-old chronic DHBV-carrier or uninfected ducks were exposed to AFB1 for 5 weeks or treated with dimethylsulfoxide (DMSO) as a control. Animals were observed for 6 to 13 weeks after AFB1 treatment to study the influence of AFB1 exposure on DHBV replication and liver pathologies. Histological analysis showed more marked changes in the livers of AFB1-treated ducks, and these were enhanced by DHBV infection. A significant increase in serum and liver DHBV DNA level was observed in AFB1-treated ducks as compared with DMSO-treated controls. In addition, viral RNAs, in particular the pregenomic RNA that is the template of viral replication, and intrahepatic DHBV DNA replicative intermediates, were significantly increased by AFB1 treatment. Moreover, an overexpression and accumulation of DHBV large envelope (L) protein was observed in the hepatocytes of AFB1-exposed animals. The in vitro study has further confirmed an increase in intracellular viral DNA and in virus release in AFB1-treated primary duck hepatocytes. Taken together, our results indicate that AFB1 exposure leads to an increase in virus gene expression associated with intrahepatic accumulation of DHBV L protein and enhanced liver pathology.     

Isolated antibodies against the core antigen of hepatitis B virus in HIV-infected patients

The aim of this study was to describe the frequency and significance of isolated antibodies against the hepatitis B virus (HBV) core antigen (HBc) in 2185 HIV-infected patients of the Aquitaine Cohort. Antibodies against HBc were found in 372 subjects (17%). Patients with isolated anti-HBc antibodies were more frequently coinfected with hepatitis C virus (HCV) (58.2%) than those who were anti-HB surface (HBs) antibody positive (22.9%, P<0.001) and those who were dually reactive anti-HBs/anti-HBc antibody positive (27.3%, P<0.001). These results suggest interactions between HBV and HCV. As observed in patients not infected with HIV, the "anti-HBc-alone" serological profile could reflect essentially late immunity with undetectable anti-HBs antibodies. However, an occult HBV infection cannot be ruled out.     

Hepatitis B virus infection and hepatocellular carcinoma: correlation between IgM antibody to hepatitis B core antigen, hepatitis B e antigen, and hepatitis B DNA

Sera from 102 black patients with primary hepatocellular carcinoma (PHC) and hepatitis B surface antigenemia were tested for immunoglobulin M antibody against hepatitis B core (IgM anti-HBc), hepatitis B e antigen (HBeAg), and hepatitis B viral (HBV) DNA. Their prevalences were compared to those of a control group of 124 age and sex matched black HBV carriers without tumor. IgM anti-HBc was present in 68.6%, HBeAg in 32.3%, and HBV-DNA in 26.7% of the patients. In the control population, IgM anti-HBc was present in 45%, HBeAg was detected in 3.2%, and HBV-DNA in 25.8%. We conclude that IgM anti-HBc is present appreciably more often than either HBeAg or HBV-DNA in patients with PHC. HBeAg or IgM anti-HBc in serum of HBsAg positive carriers may predict an added risk of PHC development in South African blacks.     

Hepatitis B virus DNA in the serum of Sardinian blood donors negative for the hepatitis B surface antigen

The high endemicity of hepatitis B virus (HBV) infection and liver disease in Sardinia led us to assess the occurrence of HBV DNA in 1,411 sera of two selected groups of hepatitis B surface antigen (HBsAg)- negative blood donors: 793 with abnormal serum alanine aminotransferase (ALT) and 618 with normal serum ALT values (determined during routine testing of their blood donation). HBV DNA sequences were detected by dot-blot hybridization in 68 of 793 subjects (9%) with abnormal ALT but only in three of 618 subjects (0.5%) with normal ALT. HBV-core antibody (anti-HBc) was detected in 338 of 793 subjects (43%) with abnormal ALT as well as in 125 of 618 subjects (20.2%) with normal ALT. Among the 71 subjects positive for serum HBV DNA, 22 (31%) were positive for anti- HBc, while 49 (69%) were negative for all serologic markers of HBV infection. Thus, a high frequency of anti-HBc in apparently healthy HBsAg-negative individuals and a high prevalence of serum HBV DNA in the absence of immunologic markers of HBV infection suggest the existence of genetic variants of HBV that may be responsible for some of the presumed NANB hepatitis encountered in Sardinia and possibly other areas of high endemicity for HBV.