Hepatitis D virus (HDV) infection causes the most severe form of viral hepatitis with rapid progression to cirrhosis, hepatic decompensation, and hepatocellular carcinoma. Although discovered > 40 years ago, little attention has been paid to this pathogen from both scientific and public communities. However, effectively combating hepatitis D requires advanced scientific knowledge and joint efforts from multi-stakeholders. In this review, we emphasized the recent advances in HDV virology, epidemiology, clinical feature, treatment, and prevention. We not only highlighted the remaining challenges but also the opportunities that can move the field forward.
Carcinoma, Hepatocellular/complications* ; Hepatitis B virus ; Hepatitis D/epidemiology* ; Hepatitis Delta Virus/genetics* ; Humans ; Liver Cirrhosis/etiology* ; Liver Neoplasms/complications*

OBJECTIVEHepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose.

METHODSReconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method.

RESULTSThe expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies.

CONCLUSIONExpression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.

Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Hepatitis D ; diagnosis ; immunology ; virology ; Hepatitis Delta Virus ; immunology ; Hepatitis delta Antigens ; immunology ; Recombinant Proteins ; genetics ; metabolism

BACKGROUNDTo construct the pRSETB-HDAg recombinant expression plasmid and to obtain soluble hepatitis D virus antigen with high biological and antigenic activity.

METHODSHDAg gene fragment was inserted into fusion expression pRSET B vector that includes T7 promoter and a polyhistidine tag. The recombinant plasmid was transformed into host bacterium BL21 after induction with IPTG. The expression supernatant was purified by chelating affinity chromatography and the recombinant HDAg antigenic activity was detected by EIA.

RESULTSEIA detection using the recombinant HDAg showed strong positive reaction with hepatitis D patients sera. The positive rates of the EIA, compared with HDAg from USA and Hua Mei EIA kit in detecting 26 cases of anti-HDV positive reference sera, were 100%, 96.15% and 100%, respectively.

CONCLUSIONRecombinant plasmid for HDAg with good antigenicity was successfully constructed and could be used as hepatitis D antibody detection reagent.

Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis Delta Virus ; genetics ; immunology ; metabolism ; Hepatitis delta Antigens ; genetics ; immunology ; metabolism ; Immunoenzyme Techniques ; Recombinant Proteins ; immunology ; metabolism

OBJECTIVETo explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.

METHODSAccording to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.

RESULTSBoth the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.

CONCLUSIONSThe cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.

DNA, Antisense ; genetics ; Genome, Viral ; Hepatitis B virus ; genetics ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; RNA, Catalytic ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Transcription, Genetic

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

OBJECTIVETo obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity.

METHODSHepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA.

RESULTSComparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistency was good (100%).

CONCLUSIONEIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.

Amino Acid Sequence ; China ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis D ; blood ; virology ; Hepatitis Delta Virus ; genetics ; immunology ; isolation & purification ; Hepatitis delta Antigens ; blood ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; immunology ; metabolism

OBJECTIVESTo investigate whether HDV ribozymes can intracellularly inhibit HCV RNA.

METHODSThe mammalian expression vectors, pC1-RzC1, pC1-RzC2 and pC1-RzC3, containing ribozymes cDNA of RzC1, RzC2, and RzC3, were constructed targeting different HCV-5' NCR-C RNA regions. Then the HCV-positive fetal hepatocytes were transfected with these plasmids using liposome-mediated method. The inhibitory effects of HDV ribozymes were evaluated by HCV RNA quantitation in cultured cells and the supernatants.

RESULTS(1) All the three HDV ribozymes were inserted into the expression vector. (2) Fetal hepatocytes were infected with HCV proven by RT-PCR and fluorescent quantitative PCR and expressed HCV NS3 and NS5 antigens by immunocytochemistry. (3) HDV ribozymes inhibited the activity of the target HCV RNA at expect positions in HCV-positive hepatocytes. At 0.5 micromol/L, the inhibitory rate of pC1-RzC1, pC1-RzC2, and pC1-RzC3 was 53.2%, 50.5 %, and 10.6% respectively. PC1-RzC1 was used continuously for one week, showing the inhibitory rate of 60.7%, 64.2%, 68.4%, 71.9%, 78.8% and 83.1% on the 2nd, 3rd, 4th, 5th, 6th and 7th day.

CONCLUSIONThe inhibitory activity of pC1-RzC1 (107-113nt) and pC1-RzC2 (268-274nt) is greater than that of pC1-RzC3 (345-351nt) in HCV-positive hepatocytes.

Genetic Therapy ; Genetic Vectors ; Hepacivirus ; drug effects ; genetics ; Hepatitis C ; drug therapy ; Hepatitis Delta Virus ; genetics ; Plasmids ; RNA, Catalytic ; therapeutic use ; RNA, Viral ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo develop HDV as a vehicle to deliver hammerhead ribozyme into hepatocytes, the effects of modified HDV was assessed on the activity of embedded hammerhead ribozyme in vitro and in vivo.

METHODSIn vitro activity of ribozyme or HDV-driven ribozyme was assessed by incubating with the [alpha-32 P]-ATP labeled HBV RNA substrates at different temperature. Huh-7 cells were cotransfected with ribozyme or HDV-ribozyme chimera and HBV genome, by which inhibition of ribozymes on HBV transcription in vivo were examined.

RESULTSThe results indicated that both temperature and secondary structure influenced the cleavage activity of HDV-driven ribozyme significantly. When the factors were eliminated, the HDV-driven ribozyme could act as well as its counterpart naked ribozyme. While in cultured cells the HDV-driven ribozyme had higher inhibition to HBV gene expression than that of ribozyme alone.

CONCLUSIONThe results demonstrated that HDV may weaken the activity of embedded ribozyme in vitro, but make it enhanced in cultured cells. Thus, this study could provide a useful evidence to develop HDV as vector for liver-special delivery of ribozyme to against chronic HBV infection.

Base Sequence ; Cell Line, Tumor ; Genome, Viral ; Hepatitis Delta Virus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Protein Structure, Secondary ; RNA, Catalytic ; chemistry ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Structure-Activity Relationship ; Substrate Specificity ; Temperature ; Transfection

BACKGROUND/AIMS: The aims of the present study were to determine the outcomes of inactive hepatitis B virus (HBV) surface antigen (HBsAg) carriers over a 10-year study period and to elucidate the HBV serological profile of their family members. METHODS: We retrospectively analyzed the medical files of inactive HBsAg carriers followed up at the Department of Infectious Diseases of Kocatepe University Medical Faculty Hospital between March 2001 and January 2011. RESULTS: In total, 438 inactive HBsAg carriers were enrolled in this trial. The follow-up period was 33.7+/-22.5 months (mean+/-SD). Anti-hepatitis-B surface antibody seroconversion occurred in 0.7% of cases, while chronic hepatitis B was found in 0.5%. The anti-hepatitis-D virus (HDV) status was evaluated in 400 patients and anti-hepatitis C virus (HCV) in 430. It was found that 1% and 0.2% were positive for anti-HDV and anti-HCV, respectively. HBV serology was investigated in at least 1 family member of 334/438 (76.3%) patients. The HBsAg positivity rate was 34.6% in 625 family members of 334 patients. A comparison of the HBsAg positivity rates in terms of HBV DNA levels in index cases revealed that HBsAg seropositivity rates were higher in family members of HBV DNA-negative patients than in family members of HBV DNA-positive cases (P=0.0001). CONCLUSIONS: The HBsAg positivity rate was higher in family members of inactive HBsAg carriers than in the general population; these family members therefore have a higher risk of HBV transmission. Furthermore, despite negative HBV DNA levels, transmission risk was not reduced in these patients, and horizontal transmission seems to be independent of the HBV DNA value.
Adult ; Antibodies/blood ; Carrier State ; DNA, Viral/analysis ; Family Health ; Female ; Follow-Up Studies ; Hepatitis B Antibodies/blood ; Hepatitis B Surface Antigens/*blood ; Hepatitis B virus/genetics/immunology ; Hepatitis B, Chronic/*diagnosis/transmission/virology ; Hepatitis Delta Virus/immunology ; Humans ; Male ; Middle Aged ; Retrospective Studies