The phylogenetic analysis of hepatitis C virus genotype 1b mutants in China / 中华检验医学杂志

Objectives In our studies before, we reported mutation C-T at -222 of hepatitis C virus type 1b genotyped on 5′ noncoding region (5′NCR). There was no this mutation of type 1b recorded in GenBank. Since genotypiog based on 5′NCR cannot differentiate subtypes, it is necessary to investigate whether the hepatitis C virus genotype 1b strains with mutation C-T belongs to other subtypes of genotype 1. Methods 64 HCV genotype 1b samples were amplified from 5′NCR by RT-PCR.Then the products were digested by use of BamHⅠ restriction enzymes. We randomly chose 6 samples with BamHⅠ restriction site and subjected them to amplification of 5′NCR and NS5B. The sequences of 5′NCR were analyzed. Sequences of NS5B fragments from the 6 samples were respectively subjected to phylogenetic analysis with subtypes of genotype 1 and 38 complete genomes of genotype 1b from GenBank.Results The phylogenetic analysis of 6 samples and subtypes of genotype 1 indicated that the genotypes of 6 strains with BamHⅠ restriction site in 5′NCR were type 1b, instead of subtype of type 1. Tree construction with 38 complete genomes of genotype 1b showed that the 6 mutants of genotype 1b did not belong to the same branch of the tree and there were no genetic differences between the mutants and the other strains of genotype 1b.Conclusions Our research indicated that the hepatitis C virus stains of C-T in 5′NCR were mutants of type 1b. Since the 5′NCR is a highly conserved region of HCV, the mutation might have some relationship to the long-time IFN therapy.

The influence of HCV core protein on cell apoptosis,cell cycles and cell telomerase activities of HepG2 cells / 中华传染病杂志

Objective To investigate the influence of HCV core protein on cell apoptosis, cell cycles and cell telomerase activities of HepG2 cells. Methods A eukaryotic expression plasmid containing HCV C gene was constructed by DNA recombinant technique and the recombinant plasmid was transfected into HepG2. Thereafter, HepG2 cells transfected with recombinant eukaryotic expression plasmid were obtained. The HCV C mRNA and protein in HepG2 cells transfected with recombinant plasmid were verified by RT PCR and indirect immunofluorescence assay. The HepG2 cells were studied as follows: (1) The cell proliferation ratio of three groups cells(HepG2 cells transfected with the recombinant plasmid, HepG2 cells transfected with blank plasmid and HepG2 cells without transfection) was evaluated by MTT assay; the cell cycles were also examined by FACS. (2) The apoptotic ratio of three groups cells were examined by FACS. (3) The cell telomerase activities of all three group cells were examined by TRAP ELISA assay. Results (1) The cell proliferation ratio in the group of HepG2 cells transfected with recombinant plasmid was higher than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection; The proportion of phase S in the group of HepG2 transfected with the recombinant plasmid was significantly higher than that of the group of HepG2 without transfection. (2) The apoptotic ratio in the group of HepG2 cells transfected with recombinant plasmid was significantly lower than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection. (3) There were no significant differences among the three group cell telomerase activities. Conclusions (1) HCV C protein had the potential role in inhibiting cell apoptosis. (2)HCV C protein could induce HepG2 cells from phase G 0/1 to phase S, and might promote cell proliferation, inhibit cell apoptosis. (3) HCV C protein had no influence on cell telomerase activities of HepG2 cells, thus HCV C protein regulated cell cycle, promoted cell proliferation and inhibited cell apoptosis not by enhancing cell telomerase activities.

The effect of hepatitis C virus core gene transfection on expression of human telomerase reverse transcriptase mRNA in uman biliary carcinoma cell lines / 中国普通外科杂志

Objective To study the effect of HCV C gene transfection on expression of hTERT mRNA in human biliary carcinoma cell lines (QBC939) and to elucidate the significance of activation of hTERT mRNA by HCV C gene on the carcinogenesis of bile duct cells. MethodsThe recombinant plasmid(pcDNA3-HCVC) and the vector-alone were co-transfected with enhanced green fluorescent protein( EGFP )into QBC939 and human normal bile duct epithelial cells(HBEC) using liposome. The reverse transcription PCR(RT-PCR) and immunocytochemical stain were used to show the expression of hTERT mRNA and protein. Results The transfection efficiency of pcDNAHCV-C,which was determined by the expression of EGFP,is about 16% and 30% in QBC939 and HBEC respectively. There was no expression of hTERT mRNA assayed in HBECs when transfected blank vector,but a dramatic increase was observed for hTERT mRNA expression in HBEC when transfected with HCV C expressing vector. The expression of hTERT mRNA and protein assayed in QBC939 significantly increased when transfected with HCV C expression vector than that transfected with blank vector. Conclusion HCV C gene transfection up-regulates the transcriptional expression of hTERT mRNA in biliary carcinoma cells,it is suggested that HCV C protein contributes to virus carcinogenesis.

Quantitation of hepatitis C virus RNA in amniotic fluid of gravida infected by hepatitis C virus / 中华妇产科杂志

Objective To detect hepatitis C virus (HCV) RNA in amniotic fluid of gravida and investigate mother-to-infant transmission of HCV. Methods Thirty-four HCV seropositive gravida (experimental group) were engaged. Fluorescence quantitative polymerase chain reaction (PCR) based on amplisensor assay and reverse transcription -PCR (RT-nPCR) was used. Serum HCV RNA positive sera were genotyped by RFLP analysis of PCR products from 5′NC region. Sera and amniotic fluid samples of 40 normal gravida were set as the control group. Results In the experimental group, HCV RNA was detected in amniotic fluid (5.9%, 2/34) of 2 cases. HCV RNA titers were 10 5 and 10 6 copy/ml respectively. No HCV RNA was detected in the amniotic fluid and sera of the control (n=40). Conclusions HCV RNA was rarely detected in amniotic fluid. The amniotic fluid is not the main route of HCV mother-to-infant transmission.

Nucleic acid amplification test of blood donations in blood bank / 中华检验医学杂志

Objectives To increase the safety of blood supply and to evaluate the feasibility of Nucleic Acid amplification test (NAT) of blood donations in blood bank. Methods Individual donor plasma samples serologically negative for HCV, HBV and HIV detected by ELISA were pooled according to the size of 20?50 ?l. HCV RNA and HBV DNA in pooled samples were detected by AcuGen AG 9600 AmpliSensor qualitative PCR methods. Individual donor plasma samples in positive pooled samples were further tested by PCR. Results One(0.01%)of 8805 donations was PCR for HCV RNA positive. Six (0.4%)of 1 441 donations were PCR for HBV DNA positive. The whole procedure took three days from pooling donor plasma samples to identifying the positive samples. Conclusion It is feasible to incorporate NAT into ELISA screening blood donations for HBV and HCV. NAT will further increase the safety of blood supply.

Two quantitative hepatitis C virus RNA reverse transcriptase PCR assays / 中华检验医学杂志

Objective To compare the real time flourescent quantitative HCV RNA PCR detective method with automated quantitative PCR (COBAS Amplicor) test. Methods The test panel consisted of 36 samples including one half ten fold dilution series of 7 samples, 20 HCV RNA positive samples, and 9 negative samples. We compared the two quantitive method, with their agreement for test results, intra assay variability, and linearity. Results The regression coefficient of linearity of COBAS Amplicor and real time flourescent quantitative method was 0.999 5 and 0.973 2, respectively. The inter assay coefficient of variation of COBAS Amplicor was lower than that of the real time flourescent quantitative method. A correlation co efficient between the HCV RNA titres as detected by the two methods was 0.845 ( P

A preliminary study on hepatitis C virus infection of a human liver cell line HepG2 in vitro / 中华传染病杂志

Objective To establish practical cell model of HCV infection, and investigate the susceptibility of a human liver cell line HepG2 to hepatitis C virus in vitro. Methods A human liver cell line HepG2 was incubated with serum from a chronic hepatitis C patient for 6～8 hours. Both the plus and minus strands of HCV RNA in infected cells or supernatant were examined by reverse transcription polymerase chain reaction (RT PCR). The HCV NS 3,NS 5 antigens in infected cells were respectively detected with the monoclonal antibodies to antigens of their own by immunohistochemical assay. The minus strand of HCV RNA in infected cells were localized by in situ hybridization. Results The intracellular plus or minus stands of HCV RNA were first detected on day 3 post incubation and then could be intermittently detected until day 35 post incubation in cells or supernatant. The positive signals of NS 3,NS 5 antigens could be expressed within cytoplasm of infected cells. The minus strand of HCV RNA was located within cytoplasm by in situ hybridization. Conclustions These results show that a human liver cell line HepG2 is not only susceptible to HCV but also able to support its long time replication in vitro.

INTRACELLULAR INHIBITION OF VIRAL GENE EXPRESSION BY HAMMERHEAD RIBOZYMES AGAINST HEPATITIS C VIRUS 5′ NCR AND C REGIONS / 解放军医学杂志

To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection,we designed and synthesized 2 hammerhead ribozymes (Rz213 and Rz498) targeting conserved sites in the 5′noncoding region (NCR) and C gene of HCV RNA.Constructed to the eukaryotic vector pcDNA3, the two ribozymes were respectively or simultaneously transfected with lipofectamine into WISHnc transgenic cells, which could express permanently HCV C luciferase protein under the control of HCV 5′NCR.The expression of C luciferase was measured by luminometer.The results showed that the luciferase activities were significantly down regulated in the WISHnc cells, and the inhibitory rates were 42.94%～67.81% within 7 days after ribozymes transfection. There was no significant differences between Rz213 or Rz498 and co transfection, but adding the target site of ribozymes might prevent host cells from the loss of ribozyme therapeutic effect due to viral gene mutation.

Study of transcription and cleavage in vitro of HCV with HCV specific hammerhead ribozyme / 中华传染病杂志

Objective To study the effect of specific hammerhead ribozyme (Rz) to hepatitis C virus (HCV) in vitro. Methods Rz 1 Rz 2 were designed to cleavage at 5′-NCR nucleotide positions under 136～160 and 313～337, Rz 3 was designed to cleavage at C region nucleotide position under 373～388. As a control, cleavage deficient Rzm that have A→G point mutations in the catalytic loop of the hammerhead domain. 32P-labeled transcript of target HCV RNA was incubated with gel-purified Rz ( Rz 1, Rz 2, Rz 3 and Rzm ) respectively at different concentration based on specified condition and autoradiographed after denaturing gel-electrophoresis. Results Except Rzm, Rz 1 Rz 2 Rz 3 were active at 37 ℃ and more so at higher concentration, and more so with cleavage site nearly to the HCV initial code. Conclusions The HCV specific hammerhead ribozyme can be designed in vitro, further study about cleavage in vitro and in vivo will continue.

Expression and purification of the fusion hepatitis C virus hypervariable region 1 protein and it's preliminary application / 中华传染病杂志

Objective To clone and express hypervariable region 1 (HVR1) fragments of different genotypes of HCV strains from different cities of China.Then detect the reactivity of the expressed HVR1 fusion proteins with sera of chronic he-patitis C patients to analyzing it's signification in clinical utilization. Methods HVR1 genes of four HCV strains (genotype 1b and 2a) were amplified from pGEMT-E2 plasmids and cloned into pQE40 vectors, respectively to construct four recombinant prokaryotic expression plasmids which expressed HVR1 as fusion proteins with DHFR in Escherichia coli strain TG1. Then we used the purified DHFR-HVR1 proteins to detect the anti-HVR1 antibodies in 70 serum samples of chronic hepatitis C patients. Results Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320～ 800 ?g fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8%(51/70), 60%(42/70), 48.6%(34/70), and 58.6%(41/30) of the anti-HCV positive patients' sera, respectively by ELISA. 57% (4/7) of the non responders' sera taken before therapy reacted with all four HVR1 fusion proteins, while only 15.3% (2/13) of the sera from responders reacted with all of them. The A values of sera from IFN therapy responders were significantly higher than non responders (P

Serum soluble IL-2 receptor levels following interferon and ribavirin combination therapy with chronic hepatitis C / 대한내과학회지

BACKGROUND: To evaluate the cellular immune response to interferon(IFN)-alpha and ribavirin combination therapy in patients with chronic hepatitis C, we monitored serum levels of soluble IL-2 receptor(sIL2R) before and after the therapy. METHODS: Serum sIL2R levels before and after the combination therapy were measured in 19 patients with chronic hepatitis C. IFN(3 MU/day, 3 times/week) and ribavirin 1000 mg/day were administered for 24 weeks to all patients. RESULTS: After the therapy, sIL2R levels were increased (before, 3.13 0.67 ng/m L, and after 4.08 2.13 ng/mL, p=0.059) but statistically insignificant(p>0.05). The patients were divided into two groups : the responder group who were negative for serum hepatitis C virus(HCV)-RNA after the therapy, and the non-responder group who were still positive for HCV-RNA after the therapy. Between these two groups, sIL2R levels before and after the therapy were not significantly different. The ratio of sIL2R levels before and after the therapy was calculated, although the ratio was higher in responder group, but there was no significant difference between the two groups(sIL2R after the therapy)/(sIL2R before the therapy) : 1.43 0.70 in the responder group and 1.04 0.28 in the nonresponder group, p=0.096). CONCLUSION: Although these results failed to demonstrate that sIL2R level was increased during the combination therapy in patients with hepatitis C, this study suggested that cytokines which mediate immune response may be involved in the pathogenesis of chronic heaptitis C virus infection.

Quantitive analysis of the inhibition of HCV IRES mediated HCV core protein expression in cells by inhibitor RNA / 中华传染病杂志

Objective To study the inhibition of HCV IRES mediated HCV core protein expression in cells by inhibitor RNA. Methods Plasmid pcRz-IRNA, a eukaryotic expression vector with IRNA and two self cleavage ribozyme overhang at both sides respectively, was constructed and co-transfected with pcHCVcluc (containing HCV NCR, core and Luc genome) into the HHCC cell line (Human Hepatocellular Carcinoma cell line). Immunoflurescence tests were applied to detect the co-transfected cells, which were thereafter analysed with confocal microscope quantitatively. Luciferase activity was valued using Luc Assay System (Promega). Results The cotransfected cells expressed HCV core protein, and the fluorecein in which was reduced significantly in comparison with control. Conclusions IRNA can inhibit the expression of HCV IRES mediated core protein in the cotransfected cells.

Cloning and expression of NS3 gene of hepatitis C virus in yeast / 中华传染病杂志

Objective HCV NS3 protein plays an important role in disease caused by HCV. We investigate the gene expression of HCV NS3 in yeast for future study of the function of the protein. Methods PCR was performed to amplify the gene of HCV NS3 from the plasmid pBRTM/HCV containing the whole fragment of HCV and the gene was cloned into pGEM T vector. Thereafter, HCV NS3 gene was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, and recombinant pGBKT7∶NS3 was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Results HCV NS3 gene was successfully cloned into pGBKT7. The results of SDS PAGE and Western blotting assay showed that the molecular weight of the expressed product was about 22000 Da and HCV NS3 protein was existed within yeast cells.Conclusions HCV NS3 was successfully expressed in yeast expression system.

Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.

Hallazgos clínicos, bioquímicos y morfológicos en 103 pacientes con anticuerpos contra el virus de la hepatitis C

Se estudiaron 103 pacientes portadores de anticuerpos contra el virus de la hepatitis C detectado mediante el empleo del sistema diagnóstico cubano (Anti VHC, CIGB, La Habana). La lesión histológica predominante fue la hepatitis crónica activa (23,2 %), aunque se presentaron 2 casos con hepatitis aguda grave por infección combinada con los virus A y B. En muchos de los enfermos la afección cursó con escasos síntomas. La mayoría de los pacientes eran donantes de sangre y receptores de transfusiones (44,5 %). Estos últimos se relacionaron con las lesiones hepáticas más severas. Se señaló la presencia de portadores sanos del virus.

103 patients carriers of hepatitis C virus antibodies detected by the Cuban diagnostic system (Anti HCV, CIGB, Havana) were studied. Active chronic hepatitis (23.2 %) was the predominant histological lesion, although there were 2 cases with acute severe hepatitis due to a combined infection with A and B viruses. A few symptoms were observed in most of the patients, who were blood donors and blood transfusion receptors (44.5 %). The latter were associated with the most severe hapatic lesions. It is stressed the presence of heal thy carriers of the virus.

Randomized,Open-label and active controlled study of PEG-IFN?-2a in the treatment of chronic hepatitis C in China / 中华传染病杂志

Objective To investigate the efficacy, tolerability and safety profiles of PEG IFN alpha 2a(PEG IFN? 2a) in the treatment of chronic hepatitis C in China. Methods 208 patients with chronic hepatitis C were included, and divided into two groups randomly, PEG IFN? 2a group and IFN? 2a Group respectively. There was no significant difference between two groups in pretreatment HCV RNA, HCV genotype and other clinical data. The main parameters to valuate the efficacy were virological response and biochemical response. The side effects were intensively observed. Results Sustained virological response rate in PEG IFN? 2a group was significantly higher than that in IFN? 2a group (41.51% and 16.67% respectively, P

Cloning and identification of human gene transactivated by hepatitis C virus core protein 1 / 解放军医学杂志

Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequences of the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. One of them was confirmed to be a new gene without homology with known genes in this database.Then, electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene, in conjunction with Kozak rule and the existence of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene with mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named TAHCCP1.The nucleotide sequence of the new gene and its corresponding protein-encoding amino acid, which was 2 001nt and composing 667aa, have been determined. The sequence of the TAHCCP1 gene has been registered in GenBank with its accession number AY038359. Conclusion TAHCCP1 gene transactivated by HCV core protein was cloned and identified successfully by a combination of molecular biological technology and bioinformatics technique. The results are expected to pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapies for chronic hepatitis C.

Preparation and immunohistochemical study of human single chain Fv antibody against hepatitis C virus core antigen / 解放军医学杂志

Objective To detect hepatitis C virus core antigen in 7721 cells transfected with HCV cDNA by immunohistochemistry method with human single chain Fv antibody(scFv). Methods The recombinant phages were panned by core antigen which was coated in a microtiter plate. After three rounds of biopanning, 48 clones were identified specific to core antigen. The affinity and specificity of scFv were evaluated by ELISA and immunohistochemistry. Results ScFv-core DNA digestion and sequence data showed that the scFv gene was composed of 774bp. Conclusion Human single chain Fv antibody against HCV core antigen has a specific combining capacity with hepatitis C virus core antigen.

Expression of soluble human anti-idiotypic single chain Fv antibody to hepatitis C core protein in E. coli / 解放军医学杂志

Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.

Screening of binding protein of hepatitis C virus NS4A by phage display from human liver cDNA library / 解放军医学杂志

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.