| Hepatitis C virus (HCV) is a serious and growing threat to human public health and is estimated to infect over 180 million people worldwide, and is highly prevalent among drug users. Hepatitis C, it had become clear that the vast majority of persons who develop acute hepatitis C, perhaps over 80 percent-remain infected. A high proportion of persons with chronic HCV infection will develope cirrhosis and progress eventually to terminal chronic liver disease, in some instances after developing hepatocellular carcinoma (HCC).Individuals infected with HCV have two possible outcomes of infection, clearance or persistent infection, determined by a complex set of virus-host interactions. The HCV genome exhibits a considerable degree of sequence variation. On the basis of these variations, HCV is now classified into at least 6 genotypes and more than 60 subtypes. HCV subtype 1b is the most prevalent in Asia, including China, and is more frequently associated with severe liver injury, the development of HCC and poor interferon teatment response than any other HCV subtype.HCV is a member of the Flaviviridae family, with a positive, single-strand RNA genome of 9.6 kb. The genome encodes a single polyprotein that is proteolytically cleaved to produce four structural (Core, E1, E2, and p7) and at least six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. The core protein is located at the N terminus of the polyprotein and is predicted to have a length of 191 amino acids and a molecular mass of 23 kDa (p23). HCV core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases.Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence. F protein is conserved in different HCV genotype, which may play an important role in the viral life cycle. The double-shift F protein (ARFP / DF) was found in HCV genotype 1b by Boulant et al in 2003. AUG codon of C gene acts as the promoter of DF, a +1 frameshift at codon 42 and, for some of them, from a rephasing in the normal open reading frame at the termination codon 144 in the -1 open reading frame. DF is a chimeric F-C protein, which is the chimeric gene product from a part of F and C gene. There is no research on the function of DF. So we carry out our work of assessing the distribution of HCV genotypes, the prevalence of serum anti-F in drug users with HCV infection, and the correlation between the double-shift F protein and chronic HCV infetion and hepatocellular carcinom cell apoptosis.Section I Influence of hepatitis C virus genotypes and anti-F protein antibodies on outcome of injecting drug users with HCV infectionObjective: To find correlates between drug users with HCV persistent infection and self-limiting infection with regard to demographic, and serological parameters. Methods: A total of 360 drug users were selected from Nanjing compulsory rehabilitation center for this study. All drug users without interferon treatment were positive for anti-HCV antibodies. According to a series of test results, 192 drug users who repeatedly lacked detectable levels of HCV RNA , and the levels of alanine aminotransferase (ALT) were under 40 U/L. These drug users were regarded to as those with a self-limiting HCV infection. For comparison reasons, the remaining 168 drug users were proven by HCV RNA in serum and the levels of ALT were all over 40 U/L. they were attributable to the HCV persistent infection group. In HCV RNA positive sera, virus genotyping was performed by using HCV Genotype Primer Kit. Sera from patients with non-detectable HCV RNA, were tested for type-specific antibodies to HCV genotypes 1, 2, 3, 4, 5, and 6 by using the MUREX HCV Serotyping 1-6 Assay. The purified recombinant protein (HCV-F /GST) was coated onto microtiter plates as antigen. The secondary antibodies were horse radish peroxidase (HRP)-conjugated goat anti-human IgG. Sera of 360 patients were tested by indirect ELISA for detecting anti-HCV-F antibodies. Results: There were significant differences in gender between the groups of patients with persistent infection and those with self-limiting infection (P=0.026). No differences in anti-HCV-F postive between the two groups were found, with the exception that anti-HCV-F postive in persistent infection was slightly higher than that in self-limiting infection (36.3% vs 27.6%). Multivariate analysis identified the following independent risk factors for HCV persistent infection: frequency of male(OR=1.43;95%CI,1.03-2.49;P=0.032), HCV genotype 1 (OR=4.18;95%CI,2.14-8.13;P=0.0001) and HCV-F antibody (OR=1.73;95%CI,1.08-2.47;P=0.047). Conclusion: This information indicates that besides HCV-F antibody, other factors including HCV genotype and sex also influence the outcome of HCV infection. Section II The double-shift F protein of Hepatitis C virus subtype 1b inhibits human hepatocellular carcinom HepG2 cell apoptosis in vitroObjective: To investigate the effects of the double-shift F protein of Hepatitis C virus subtype 1b on the apoptosis of human hepatocellular carcinom HepG2 cells. Methods: HepG2 cells were transfected with recombinant plasmid pcDNA3.0-DF-EGFP. And pcDNA3.0-DF-EGFP-HepG2 strain was exposed to Act-D and tumor necrosis factorα(TNFα) treatment to induce cell apoptosis with positive control pcDNA3.0-C-EGFP-HepG2, negative control pcDNA3.0-C-EGFP- HepG2 and blank control HepG2. Annexin V-FITC/PI of Flow cytometry was performed to determine the number of apoptotic cells. DNA Ladder was used to observe the isolation of apoptotic DNA fragments in the apoptotic cells. Results: pcDNA3.0-DF-EGFP- HepG2 cell strain showed a much delayed apoptosis as well as obviously lowered apoptotic rate in comparison with the pcDNA3.0-HepG2 strain and HepG2 strain (P<0.001) . Conclusion:The introduction and expression of extraneous gene (the double-shift F gene of Hepatitis C virus subtype 1b ) significantly inhibits the apoptosis of HepG2 cells.
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