OBJECTIVES: The seroprevalence of hepatitis E virus (HEV) among high-risk groups overseas is high, but studies in these groups are rare in South Korea. We conducted the present study from April to November 2012 to obtain data on the seroprevalence and associated risk factors for HEV among slaughterhouse workers in South Korea. METHODS: Slaughterhouse workers from 80 workplaces nationwide were surveyed in South Korea in 2012. The subjects comprised 1848 cases: 1434 slaughter workers and 414 residual products handlers. By visiting 80 slaughterhouses, which were mixed with 75 of which also performed residual products handling, we conducted a questionnaire survey for risk factors and obtained blood samples in order to determine the seropositivity and seroprevalence of HEV. Anti-HEV IgG and IgM were measured using HEV IgG and IgM enzyme-linked immunospecific assay kits and HEV antigen was measured by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The seropositivity of anti-HEV IgG was 33.5% (slaughter workers 32.8% and residual products handlers 36.2%), and among the seropositive individuals the seroprevalence of anti-HEV IgM was 0.5% (slaughter workers 0.5%, residual products handlers 0.7%). The response rate of HEV-antigen as measured by RT-PCR was 0.2%. Risk factors significantly related to anti-HEV IgG seropositivity were age, sex , and working duration (slaughter workers only). CONCLUSIONS: There were significant risk factors (sex, age, and working duration) for HEV identified in our study. All three positive cases for HEV-antigen by RT-PCR were related to pig slaughter but without statistical significance. To prevent HEV, an educational program and working guidelines may be needed for high risk groups.
Abattoirs ; Adult ; Aged ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis Antibodies/blood ; Hepatitis E/*diagnosis/epidemiology/virology ; Hepatitis E virus/genetics/*immunology/metabolism ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Male ; Middle Aged ; Multivariate Analysis ; Prevalence ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction ; Risk Factors ; Workplace

BACKGROUNDSome hepatitis B extracellular antigen (HBeAg)-positive chronic hepatitis B (CHB) patients in their immune active phase can clear the virus spontaneously and enter into an inactive hepatitis B virus (HBV) carrier state, indicating a benign prognosis. In this study, the association between cytokine-inducible SRC homology 2 domain protein (CISH) gene polymorphisms at -292 (rs414171) and the spontaneous clearance of HBV in HBeAg-positive CHB patients in immune the active phase was investigated.

METHODSSeventy HBeAg-positive CHB patients in the immune active phase were followed up for 76 weeks without antiviral therapy. The alanine transaminase, aspartate transaminase, HBV DNA, HBeAg and hepatitis B extracellular antibody levels were tested regularly. At week 76, 27 patients were classified into group A (HBV DNA level below 2 104 IU/ml and the value of HBeAg declined below 10% of the baseline at week 76), and 43 patients were classified into group B (HBV DNA level higher than 2×10(4) IU/ml or the value of HBeAg did not decline substantially at week 76). CISH (rs414171) polymorphisms were also tested using the iPLEX system.

RESULTSThe HBV DNA levels at week 12 were significantly greater in group B compared with group A (group A: (6.87±1.40) log10IU/ml; group B: (7.61±1.38) log10IU/ml, P = 0.034) and the HBeAg values were greater in group B at week 28 compared with group A (P = 0.001). The differences in HBV DNA and HBeAg values increased between the groups over time. Sixteen patients in group A and 11 in group B were genotype AA. Those with genotype AT or TT included 11 in group A and 31 in group B (AA vs. AT and TT, odds ratio 4.10 (95% confidence interval: 1.462-11.491), P = 0.006).

CONCLUSIONCISH gene polymorphisms at -292 (rs414171) are associated with HBV clearance in HBeAg-positive CHB patients in the immune active phase, and AA is a favorable genotype for this effect.

Adolescent ; Adult ; Aged ; Female ; Genotype ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Suppressor of Cytokine Signaling Proteins ; genetics ; Young Adult

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.
Animals ; Antibodies, Anti-Idiotypic/*analysis/blood/genetics ; Capsid Proteins/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Hepatitis E/diagnosis/immunology/*veterinary/virology ; Hepatitis E virus/genetics/*isolation & purification/metabolism ; Immunoglobulin G/blood/genetics ; ROC Curve ; Recombinant Proteins/genetics/metabolism ; Swine ; Swine Diseases/*diagnosis/immunology/virology

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.
Animals ; Dependovirus ; genetics ; metabolism ; Disease Models, Animal ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; physiology ; Hepatocytes ; immunology ; virology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transduction, Genetic ; Virus Replication

OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo study PD-1 and PD-L1 expressions during 24 weeks telbivudine antiviral treatment in patients with chronic hepatitis B (CHB) and to explore the relationship between PD-1 expression and HBeAg/HBeAb seroconversion.

METHODSTen CHB cases with HLA-A2 and HBeAg positive were treated with telbivudine 600 mg/d orally for 24 weeks. Fresh blood samples were collected at week 0, 12 and 24 after treatment. HBV-specific CD8+ T cells were expanded in vitro. Cell culture medium were collected for interferon gamma (IFNgamma) detection. Flow cytometry was used to detect the HLA-A type, PD-1, PD-L1 and HBV specific CD8+ T cells. The expressions of PD-1 and PD-L1, the counts of HBV-specific CD8+ T cells in circulating CD8+ lymphocytes, and IFNgamma concentration in culture medium were evaluated during antiviral treatment.

RESULTSAt week 0, 12 and 24 after telbivudine treatment, 7 of 10 patients were HBV DNA undetectable, 2 were HBeAg seroconversion and 2 were HBeAg lose but anti-HBe negative. The frequency of PD-1-positive PBMCs were 52.1%+/-17.0%, 39.1%+/-18.2% and 23.4%+/-16.3% (week 24 vs week 0, P < 0.01) respectively; PD-L1 positive PBMCs were 45.6%+/-15.4%, 34.6%+/-16.2% and 20.9%+/-9.5% respectively(week 24 vs week 0, P < 0.01; week 24 vs week 12, P < 0.05). The frequency of PD-1-positive CD8+ T cells were 76.2%+/-10.4%, 66.5%+/-15.4% and 49.5%+/-25.3% respectively (week 24 vs week 0, P < 0.01; week 12 vs week 0, P < 0.05; week 24 vs week 12, P < 0.05); HBV-specific CD8 cells were 1.3%+/-0.5%, 1.5%+/-1.0% and 2.2%+/-1.5%; IFNgamma levels in cell culture medium were (91.7+/-82.1) pg/ml, (99.4+/-93.5) pg/ml and (109.5+/-86.6) pg/ml. A remarkable decrease of PD-1 and PD-L1 expressions and increase of HBV-specific CD8+ T cells were observed in patients who had HBeAg/HBeAb seroconversion at week 24.

CONCLUSIONSDirect suppression of HBV replication by telbivudine in CHB patients can decrease PD-1 and PD-L1 expressions and restore HBV-specific CD8+T cells. The relationship between the changes of PD-1 expression and HBeAg/HBeAb seroconversion during antiviral therapy in HBeAg-positive patients need to confirm by future study.

Adult ; Antiviral Agents ; therapeutic use ; B7-H1 Antigen ; metabolism ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; drug therapy ; metabolism ; Humans ; Male ; Nucleosides ; therapeutic use ; Programmed Cell Death 1 Receptor ; metabolism ; Pyrimidinones ; therapeutic use ; Thymidine ; analogs & derivatives ; Young Adult

OBJECTIVEo study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.

METHODSHepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.

RESULTSHBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.

CONCLUSIONAd-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.

Adenoviridae ; genetics ; Calibration ; Cell Line, Tumor ; DNA, Complementary ; genetics ; metabolism ; DNA, Viral ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; metabolism ; physiology ; Humans ; Polymerase Chain Reaction ; Time Factors ; Transfection ; Virus Replication

Hepatitis E, an acute infectious disease transmitted via the fecal-oral route, is caused by hepatitis E virus. However, no effective treatment currently exists for hepatitis E, and the only epidemic control approach is vaccination. But so for there are no commercial vaccine for hepatitis E available in the world. To find a new expression system to develop recombinant hepatitis E vaccine, in this study the expression system of methylotrophic yeast Hansenula polymorpha was used to express the gene encoding amino acid 112 - 607 of the open reading frame 2 (ORF2) of hepatitis E virus (HEV) genotype IV. In order to achieve high expression level, the coding sequence was optimized according to codon usage bias of Hansenula polymorpha and synthesized through overlapping PCR. Subsequently the gene was subcloned into the multi-copy expression vectors of Hansenula polymorpha, which include pDGXHP1.0 (MOX promotor), pDGXHP2.0 (MOX promotor) and pDGXHP2.1 ( FMD promotor). The series of one-copy and multi-copy recombinant plasmids were transformed into ATCC26012(Ura3-) by electroporation. The transformants were cultured in selection media MDL and screened for the existence of foreign gene by PCR. Then the strains were induced in MM media and the expression products were detected by SDS-PAGE, ELISA and Western blot assays to select the high-level expression strains. The result of SDS-PAGE showed that the HEV ORF2 expression product was accumulated up to 12% of total cellular protein and its molecular weight is 56kD. The expression product showed high immunoreactivity detected by ELISA and the highest titer is 1:2048. The result of Western blot demonstrated that the expression product could be specifically recognized by the polyclonal antibody against HEV. The successful expression of HEV ORF2 protein in Hansenula polymorpha provides foundation for the further development of recombinant subunit vaccine against hepatitis E.
Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Genotype ; Hepatitis E ; immunology ; virology ; Hepatitis E virus ; genetics ; immunology ; metabolism ; Humans ; Pichia ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; metabolism ; Viral Hepatitis Vaccines ; immunology ; Viral Proteins ; genetics ; immunology ; metabolism

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