Establishing a new animal model for hepadnaviral infection: susceptibility of Chinese Marmota-species to woodchuck hepatitis virus infection.

Hepatitis B virus infection (HBV) is a major medical problem in China. The lack of a suitable infection model in China is recognized as an obstacle for research on HBV in China. Chinese Marmota-species is phylogenetically closely related to Marmota monax, thus, it might be suitable to serve as an animal model for HBV infection. Therefore, we attempted to prove the claim about the existence of woodchuck hepatitis virus (WHV)-like viruses in Chinese Marmota-species and to determine the susceptibility of these species to experimental WHV infection. In the present study, 653 sera from three Chinese Marmota-species, Marmota himalayana, Marmota baibacina and Marmota bobak, were screened for WHV-like viruses by serological and molecular assays. The susceptibility to WHV of three species was investigated by experimental infection and monitored by testing of anti-WHc and WHsAg by ELISA, detection of WHV DNA by PCR, and detection of WHV replication intermediates and antigens in liver samples. No evidence for the existence of a genetically closely related virus to WHV in three Chinese Marmota-species was found by serological assays and PCR. M. himalayana was susceptible to WHV infection as inoculated animals became positive for anti-WHc, WHsAg and WHV DNA. Further, WHV replication intermediates and proteins were detected in liver samples. In contrast, M. baibacina remained negative for tested virological parameters. M. bobak species showed a limited susceptibility to WHV. Our data do not support early reports about WHV-like viruses in China. M. himalayana is suitable for the establishment of a model for hepadnaviral infection.

[The immunization effects of HBV core antigen and surface antigen fusion protein in mice].

OBJECTIVE: To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein. METHODS: The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined. RESULTS: The plasmid containing the chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also caused significant HBcAg specific lymphocyte proliferation. Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg can induce more effective cellular response but weaker humoral response. CONCLUSION: Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.

[S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients].

OBJECTIVES: To investigate S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients. METHODS: HBV S gene from 8 patients (Group A) with HBsAg (+)/HBsAb (+) and 9 patients (Group B) with HBsAg (+)/HBsAb (-)was amplified by polymerase chain reaction (PCR) and sequencing. Both the distribution of genotype and serotype and the rate of MHR region were compared by Fisher's exact test. The mutation rate of both the DNA level and amino acid level was compared by t test. RESULTS: No significant difference in distribution of genotypes was found between the two groups (P=0.153). In group A, 2 were genotype B, 6 were genotype C; In group B, 6 were genotype B, 3 were genotype C. No significant difference in distribution of serotypes was found between the two groups, either (P=0.218). In group A, 2 were adw, 5 were adr, 1 was ayr; In group B, 6 were adw, 3 were adr. The mutation rate of Pre-S1 region at both the DNA level (2.29% vs 1.80%, t=2.66, P more than 0.05) and the amino acid level (2.66% vs 1.59%, t=1.39, P>0.05) was not significantly different between these two groups; the mutation rate of Pre-S2 region in group A patients was significantly higher than that in group B at the DNA level (1.74% vs 0.91%, t=4.68, P<0.01), but not higher at the amino acid level (3.18% vs 2.05%, t=1.85, P>0.05), the mutation rate of S region in group A patients was significantly higher than that in group B at both the DNA level (2.13% vs 0.81%, t=6.00, P<0.01) and the amino acid level (4.37% vs 1.52%, t=5.32, P<0.01). Amino acid substitutions were found both within and beyond the MHR region. The rate of "a" determinant mutations in these two groups was also found to be significantly different (P<0.05). CONCLUSION: Higher HBV S gene mutation rate exists in HBsAg/HBsAb double positive patients than that in HBsAg (+)/HBsAb (-) patients.

[Expressions of phosphorylated-Smad2 and PTEN in hepatocellular carcinomas and adjacent liver tissues].

OBJECTIVE: To investigate the expressions of phosphorylated Smad2 (P-Smad2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in hepatocellular carcinoma (HCC) tissues. METHODS: The expressions of P-Smad2 and PTEN were detected using Envision immunohistochemical technique in 31 cases of HCC tissues, 25 cases of HCC adjacent liver tissues and 13 cases of non-hepatocellular carcinoma tissues. RESULTS: The positive expression and staining intensity of PTEN in the cytoplasm of HCC cells (64.5%, 4.19+/-3.31) was significantly lower than those of the cells of the cancer adjacent tissues and non-cancerous tissues (96.0%, 7.88+/-0.93; 100%, 7.77+/-0.93). The staining intensity of PTEN in the cytoplasm of Edmondson pathologic grade III HCC cells was lower than those of the Edmondson grade I. The expression of PTEN was negatively correlated with intrahepatic vascular cancer thrombi (r=-0.43) and the expression of PTEN in the nuclei or cytoplasm of liver cells was negatively correlated with the liver disease progressions (r=-0.34). The positive rate and expression intensity of phosphorylated Smad2 in nuclei of HCC cells were the same as those in cancer adjacent and non-tumor liver tissues. The expression was mostly in the nucleus and cytoplasm of Edmondson grade I HCC cells, cancer adjacent liver tissue cells and non-tumor liver tissues, but its expression was only in the nuclei of Edmondson grade II and III HCC cells. The phosphorylated Smad2 expression appeared in the nuclei and in the cytoplasm of liver cells and it was positively correlated with the severity of the tumor pathology (r=0.22). Spearman correlation analysis revealed a significant inverse correlation between PTEN and phosphorylated Smad2 in HCC tissues (r=-0.73). CONCLUSIONS: The aberrant expressions of PTEN and phosphorylated Smad2 and their interaction may play an important role in the pathogenesis of hepatocellular carcinoma.

[Common linear B-cell epitopes in human hepatitis B virus core protein and woodchuck hepatitis virus core protein].

OBJECTIVE: To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus. METHODS: Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared. RESULTS: Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively. CONCLUSION: The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.

[The growth inhibition effects of TSLC1 gene on human hepatocyte carcinoma cell line HepG2].

OBJECTIVES: To study the effects of tumor suppressor in lung cancer-1 (TSLC1) on human hepatocyte carcinoma cell line HepG2. METHODS: A full length of TSLC1 cDNA was amplified from RNA of normal human liver cells by RT-PCR, and it was cloned into a pCI-neo expression vector and transfected into human hepatocellular carcinoma cell line HepG2. The HepG2 cells transfected with this plasmid (experimental group) and those treated with pCI-neo vector (control group) and without any treatment (blank group) were compared. Cell morphology was studied microscopically and cell growth was analyzed with MTT assay. FACSort flow cytometry analysis was performed to assess the cell cycle distribution and apoptosis. RESULTS: A stable cell line expressing TSLC1 protein was successfully established. Morphologically, cells of the experimental group were tightly aggregated when compared with those of the control and blank groups. The growth of TSLC1-transfected cells was significantly suppressed in vitro compared with those of the control and blank groups. The amount of G0-G1 cells was 63.66%+/-3.83% (P less than 0.01) in the experimental group, while those of the control and blank groups were 47.45%+/-0.91% and 54.47%+/-0.96% respectively. The amount of S phase cells in the experimental group, 22.90%+/-6.04%, was significantly lower (P less than 0.05) than that of the control group (36.58%+/-0.61%) and the blank group (33.61%+/-2.99%), which suggested a G0-G1 cell cycle arresting. The number of cells in early and late phase apoptosis (17.09%+/-0.20% and 16.11%+/-0.40% respectively) were significantly higher than those of the control and blank groups (P less than 0.01). CONCLUSIONS: TSLC1 strongly inhibits the growth of HepG2 cells in vitro and induces apoptosis of the cells, suggesting that TSLC1 may have a tumor suppressor function in HCC.

[Antigenicity of major hydrophilic region II of hepatitis B virus surface antigen].

OBJECTIVE: To study whether the substitutions at the major hydrophilic region II (MHRII) of hepatitis B surface antigen (HBsAg) will impair the antigenicity of HBsAg. METHODS: Four recombinant plasmids expressing mutant HBsAg (mtHBsAg) P1120T, C121S, K122I and T123N were constructed. HepG2 cells were transfected with the four plasmids and a plasmid expressing G145R HBsAg. The immunoreactivity of the cells expressing mtHBsAg with P1120T, C121S, K122I, T123N and G145R were detected by immunofluorescence (IF) staining and ELISA with 4 antibodies and 7 HBsAg diagnostic kits respectively. RESULTS: mtHBsAg with P120T was recognized by mAb1 and mAb2. mtHBsAg with C121S and K122I was not recognized by any mAbs. mtHBsAg with T123N in lysates was recognized by mAb2, but not recognized in the supernatants. CONCLUSION: Substitutions at amino acid positions 120-123 of HBsAg strongly impaired the antigenicity of HBsAg, a fact that was not appreciated previously.

[Prokaryotice expression of the NS1 gene of PPV and renaturation of the recombinant protein].

The antigen of NS1 gene of PPV was amplified by PCR, and the amplified fragments were cloned into the prokaryotic expression vector pGEX-4T-1. The insert position, the size and the frame were identified by PCR, restriction enzyme digestion and the sequence analysis of the recombinant plasmids. The sequence analysis results of pGEX-NS1-HN1 showed that the prokaryotic expression vector was successfully constructed. The target gene was successfully expressed in the host cell BL21 when induced with IPTG. The expression was optimized with proper inducing conditions of 1.0mmol/L IPTG, 10 hours and 37 degree C induction. The expression of the target protein added up to 29.8% of the total bacterial protein. The results of SDS-PAGE indicated that molecular weight of the expressed protein was about 52kDa and the expressed protein mainly existed in the inclusion body. Western blot analysis proved the recombinant protein has good reactive ability against PPV positive serum. The pGEX-NS1-HN1 inclusion body was dissolved with 8mol/L urea. Then the expressed protein was renatured by dilution method and the systems of GSH and GSSG. ELISA detection proved the renaturation protein has good biological activity.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

[Inhibition of hepatitis B and duck hepatitis B virus replication by APOBEC3G].

OBJECTIVE: To 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). METHODS: Total 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. RESULTS: CMV-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. CONCLUSION: APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

[Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma].

OBJECTIVES: To construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR. METHODS: HCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS: Recombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSIONS: The truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.

[Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells].

OBJECTIVE: To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells. METHODS: Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity. RESULTS: The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction. CONCLUSION: The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.

[Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance].

OBJECTIVE: To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance. METHODS: Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers. RESULTS: A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group. CONCLUSION: The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.

[Effect of climate change on the fisheries conununity pattern in the overwintering ground of open waters of northern East China Sea].

Data sets of 26 fisheries target species from the fishery-depen-dent and fishery-independent surveys in the overwintering ground of open waters of northern East China Sea (OW-NECS), combined sea surface temperature (SST), were used to examine the links between diversity index, pattern of common variability and climate changes based on the principal component analysis (PCA) and generalized additive model (GAM). The results showed that the shift from a cold regime to a warm regime was detected in SST during the 1970s-2011 with step changes around 1982/ 1983. SST increased during the cold regime and the warm regime before 1998 (warming trend period, 1972-1998), and decreased during the warm regime after 1998 (cooling trend period, 1999-2011). Shannon diversity index was largely dependent on the filefish, which contributed up to 50% of the total production as a single species, with low diversity in the waters of the OW-NECS, during the late 1980s and early 1990s. Excluding the filefish, the diversity index linearly increased and decreased during 1972-1998 and 1999-2011, respectively. The variation pattern generally corresponds with the trend in water temperature, strongly suggesting the effect of the SST on the diversity. The first two components (PC1 and PC2) of PCA for target species, which accounted for 32.43% of the total variance, showed evident decadal variation patterns with a step change during 1992-1999 and inter-annual variability with short-period fluctuation, respectively. It seems that PC1 was associated with large scale climatic change, while PC2 was related to inter-annual oceanographic variability such as ENSO events. Linear fitting results showed winEOF1 had significant effect on PC1, and GAM analysis for PC1 showed that winter EOF1 (winEOF1) and summer EOF2 (sumEOF2) can explain 88.9% of the total variance. Nonlinear effect was also found between PC2 and win EOF1, indicating that the fish community structure, which had predominantly decadal/inter-annual variation patterns, was influenced by inter-annual variations in oceanographic conditions.

[Prokaryotic expression and polyclonal antibody preparation of truncated duck hepatitis B virus core protein].

AIM: To construct a prokaryotic plasmid expressing truncated duck hepatitis B virus core protein (DHBc(1-214)), purify the recombinant protein, and to develop polyclonal antibodies against DHBc. METHODS: DHBc(1-214) was cloned into vector pRSET-B, then expressed in E.coli Rosetta(DE3) pLacI induced by IPTG. The recombinant protein was purified using Ni-NTA spin column. Polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS: Recombinant plasmid expressing truncated DHBc(1-214) was successfully constructed. A protein of 28,000 was expressed and purified. Polyclonal serum antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSION: The truncated recombinant DHBc(1-214) developed in this study is purified and shown strong antigenecity. The polyclonal antibody against DHBc protein is generated by regular immunization method, demonstrating both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of DHBV.