OBJECTIVETo clarify the relationship between fatal severe form hepatitis occurred during chronic hepatitis B and superinfections of hepatitis A, C, D or E virus as well as hepatitis B e system status and to adopt corresponding measures to reduce the mortality of chronic hepatitis B.

METHODSThis study detected the superinfections with hepatitis A, C, D or E virus and hepatitis B e system status in 219 patients with fatal severe form hepatitis occurred during chronic hepatitis B by enzyme linked immunosorbent assay.

RESULTSThe superinfections with hepatitis A, C, D or E virus were found in 1.4% (3/219), 9.6% (21/219), 1.8% (4/219) and 30.1% (66/219) of the patients, respectively, altogether 42.9% (94/219); hepatitis E was prominent and steady in superinfection rate in recent ten years. The causes of 57.1% (125/219) patients were not clear. The positive rate of HBeAg and anti-HBe were 17.0% (16/94) and 54.2% (51/94) in the group of superinfections with hepatitis A, C, D or E virus; and were 27.2% (34/125) and 47.2% (59/125) in the group with unknown causes, respectively.

CONCLUSIONThese results suggested that the patients with superinfections reached 42.9% (94/219), and the superinfections may be a part of causes of fatal severe form hepatitis, and the mortality of chronic hepatitis B may be decreased by strict food sanitation and use of safe blood products. There were no significant relation between hepatitis B e antigen seroconversion and the fatal severe form hepatitis occurred during chronic hepatitis B.

Adult ; DNA, Viral ; blood ; genetics ; Female ; Hepacivirus ; genetics ; physiology ; Hepatitis A virus ; genetics ; physiology ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; immunology ; physiology ; Hepatitis B, Chronic ; blood ; mortality ; virology ; Hepatitis Delta Virus ; genetics ; physiology ; Hepatitis E virus ; genetics ; physiology ; Host-Pathogen Interactions ; Humans ; Male ; Middle Aged ; Superinfection ; virology ; Survival Rate

Hepatitis E outbreaks are a serious public health concern in developing countries. The disease causes acute infections, primarily in young adults. The mortality rate is approximately 2%; however, it can exceed 20% in pregnant women in some regions in India. The causative agent, hepatitis E virus (HEV), has been isolated from several animal species, including pigs. HEV genotypes 3 and 4 have been isolated from both humans and animals, and are recognized as zoonotic pathogens. Seroprevalence studies in animals and humans indirectly suggest that HEV infections occur worldwide. The virus is primarily transmitted to humans via undercooked animal meats in developed countries. Moreover, transfusion- and transplantation-mediated HEV infections have recently been reported. This review summarizes the general characteristics of hepatitis E, HEV infection status in animals and humans, the zoonotic transmission modes of HEV, and HEV vaccine development status.
Animals ; Genotype ; Hepatitis E/*epidemiology/mortality/*transmission/virology ; Hepatitis E virus/genetics/*physiology ; Humans ; Zoonoses/epidemiology/mortality/*transmission/virology

To investigate the expression and localization of various functional domains of ORF1 polyprotein and ORF3 protein of hepatitis E virus in host cells, the coding sequences of the various functional domains (RdRp, HEL, MET, PLP, X) of ORF1 were separately cloned into pcDNA3. 1-GFP vectors for constructing the recombinant plasmids which were verified by enzyme digestion and sequencing. The exact expression of the fusion proteins were detected by Western Blot, and the distribution and localization were observed by the laser scanning confocal microscope(LSCM). In huh7 cells, GFP-RdRp proteins were found mainly in the nuclei, GFP-HEL proteins were distributed vesicularly around the nucleus, GFP-MET proteins were distributed granularly both in the nuclei and the cytoplasm, GFP-PLP proteins had polar distribution around the nucleus, and unknown GFP-X proteins were distributed uniformly both in the nuclei and the cytoplasm. Different localization of these proteins verified the previous data obtained from in vitro studies, providing a support for further research on the biological functions of various proteins coded by HEV genome.
Blotting, Western ; Cells, Cultured ; Hepatitis E virus ; genetics ; Humans ; Open Reading Frames ; Viral Proteins ; genetics ; physiology

Hepatitis E, caused by hepatitis E virus (HEV) infection, usually leads to an acute clinical course, and is the most common diagnosis among cases of acute viral hepatitis. From 2008, there have been increasing reports of chronic HEV infection in immunocompromised patients such as organ transplant recipients. Without intervention with antiviral treatment, approximately 60% of HEV infections in organ transplant recipients evolve into chronic HEV infections. Of these chronic hepatitis E patients, 10% may develop liver fibrosis and progress to liver cirrhosis. This article reviews chronic HEV infection and treatment in organ transplant recipients.
Animals ; Antiviral Agents ; therapeutic use ; Hepatitis E ; drug therapy ; virology ; Hepatitis E virus ; genetics ; isolation & purification ; physiology ; Hepatitis, Chronic ; drug therapy ; virology ; Humans ; Transplant Recipients ; Transplants ; virology

OBJECTIVETo investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.

RESULTSCMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.

CONCLUSIONAPOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

APOBEC-3G Deaminase ; Cytidine Deaminase ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B Virus, Duck ; physiology ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; RNA, Messenger ; genetics ; Virus Replication

OBJECTIVETo evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism.

METHODSA eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR.

RESULTSThe HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication.

CONCLUSIONHSP90 can inhibit HBV replication and TBK1 is not involved in this process.

HSP90 Heat-Shock Proteins ; genetics ; Hep G2 Cells ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Transfection ; Virus Replication

OBJECTIVETo establish a highly expressing and replicating hepatitis B virus (HBV) genome transgenic mouse models for screening anti-HBV drugs and investigating the pathogenesis of hepatitis B.

METHODSElongated HBV genome as the investigated gene was transducted into the pronuclei of the fertilized eggs of mice by the technique of microinjection, then the eggs were transplanted into the oviducts of the pseudopregnant mice. All the newborn mice were screened and identified by PCR and Southern blot detecting genomic DNA in tail tissue, then the positive mice were examined plasma HBsAg, HBeAg by ELISA and plasma HBV DNA by Southern blot.

RESULTSAmong the 61 offsprings, 18 were positive for tail tissue HBV DNA examination, 7 of which were positive for replication and expression detection.

CONCLUSIONTransgenic mice with elongated HBV genome possess high efficiency of replication and expression, which can be used for further investigation.

Animals ; DNA Replication ; DNA, Viral ; genetics ; Disease Models, Animal ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B e Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Mice ; Mice, Transgenic ; genetics ; Virus Replication

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects ; Trans-Activators/*antagonists & inhibitors/immunology ; Immunoglobulin Variable Region/genetics/metabolism/*pharmacology ; Hepatitis B virus/*drug effects/physiology ; Hepatitis B e Antigens/metabolism ; Cell Line

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration

OBJECTIVESTo evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro.

METHODS(1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry.

RESULTSOur data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment.

CONCLUSIONOur results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; DNA, Viral ; biosynthesis ; Flow Cytometry ; Gene Expression Regulation, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; RNA, Small Interfering ; genetics ; metabolism ; Virus Replication ; genetics