| Backgrounds and AimsHepatitis C virus (HCV) is a major etiological agent of chronic hepatitis, and among these.20%to30%will gradually develop into liver cirrhosis and hepatocellular carcinoma (HCC) in10to30years of the infection. It has been estimated that there are180million patients worldwide, of whom most are infected with HCV. The high persistence of HCV infections, together with the low sensitivity to antiviral treatments, is probably due to a complex interaction between the genetic diversity of the virus and the host immune response. The combination of pegylated interferon (IFN)-a and ribavirin is the approved and well accepted for chronic hepatitis C but it fails to cure about50%of treated patients. In patients infected with HCV genotype1b, SVR rates after standard-of-care are on the order of15%-50%. The SVR rates are considerably higher in patients infected with HCV genotypes2and3, on the order of80%. Genotype1b is by far the most prevalent subtype in China, and at the same time, we should notice that even infected with the same genotype, lb for instance, the treatment outcomes would be rather different. Two protease inhibitors, telaprevir and boceprevir have recently been be approved by FDA, but they still need further studies. And because HCV infection has chronic and hidden features, with the progression, the number of hepatitis C related end-stage liver disease patients will continue to grow in next two decades, causing a huge burden of disease. Why only parts of the patients infected with lb genotype are likely to have a favorable response to interferon treatment? Which factors were correlated with the treatment outcome of the patients infected with HCV? How to treat the patients with IFN more effective and more rational and to improve the efficacy of HCV antiviral therapy? It is a hot and difficult issue in chronic hepatitis C antiviral therapy research.Hepatitis C virus belongs to the genus hepacivirus in the family Flaviviridae. The viral genome of HCV is characterized by a single positive strand RNA with a nucleotide length of9.6kb containing an open reading frame (ORF) with untranslated regions at both ends. HCV RNA encodes a polyprotein composed of about3000amino acids. This polyprotein is cleaved by host and viral proteases into structural and non-structural proteins The core protein, and two envelope proteins are classified as structural protein, while NS2, NS3, NS4A, NS4B, NS5A, and NS5B are non-structural proteins, which play an important role in HCV life cycle, cell signal transduction, polyprotein translation and RNA replication. HCV is highly diverse genetically, with six major genotypes differing from each other by approximately30%-35%at the nucleotide level. The variability of HCV genome and lack of cell culture system and small animal model supporting HCV Production are the major impediments in HCV research and anti-HCV drug and vaccine development.A major step forward was the development of the replicon system in1999. The first generation functional HCV replicon was established. It is a bicistronic replicon and comprised:(i) the HCV5’UTR and the first12codons of the capsid protein fused in-frame with the selectable marker gene, neomycin phosphotransferase (Neo), which upon expression confers resistance to the cytotoxic drug G418;(ii) the IRES element from encephalomyocarditis virus (EMCV), which directs translation of the HCV non-structural proteins; and (iii) the HCV3’UTR. Transfection of Huh-7cells with transcripts synthesized in vitro and selection with G418resulted in a low number of surviving cell colonies. HCV replicon usually replicate poorly in cell culture at the very beginning, But it will replicate at high levels after a series of passage. Analysis of the viral sequences in these so called replicon cell lines revealed the presence of a variety of adaptive mutations, which are necessary for efficient RNA replication in cell culture. These mutations were scattered throughout the NS3-to-NS5B coding region, but most of them cluster in the central region of NS5A. And mutations in the one position will increase RNA replicons cooperarively when combined with other mutations.Adaptive mutation is one of the key to establish the cell culture system successfully. It will increase RNA replication by affect directly or indirectly interactions with host cell factors. Followed by other cell culture systems harboring the replicon of different HCV genotype, such as HCV1a (H77),2a (JFH1). The replicon system is instrumental to identify host cell factors required for RNA translation and replication and to study the mechanism that is operating in regulating the switch from translation to replication and vice versa. This system can be used for screening the compounds for antiviral activity, especially the NS3and NS4proteanes inhibitors. In spite of these achievements, we are still faced with several limitations that need to be overcome in order to make the system more applicable for HCV research. Therefore, many researchers committed to setup a cell culture system which can produce infectious HCV virus particles.In2005, Wakita T, Zhong J and Lindenbach BD et al. setup the infectious HCV genotype2a cell culture system successfully, respectively. The robust HCV cell culture infection system based on the HCV JFH-1molecular clone allows the production of virus that can be efficiently propagated in tissue culture. As a breakthrough, the cell cultures technology has made rapid development and is widely used in research, but we are still faced with several limitations that need to be overcome. Until now, the efficient cell culture systems established were all on the basis of JFH1, but genotype1b is by far the most prevalent subtype in China. There are many difficulties to setup a HCV lb cell culture system, but the researchers have made great efforts. In2005, Heller T and his team constructed an infectious HCV genotype1b cDNA between two ribozymes that are designed to generate the exact5’ and3’ends of HCV. This system supports the production and secretion of high-level HCV virions but with a low infectivity, Eventhough, it extends the repertoire of tools available for the study of HCV biology. The researchers transfected the plasmid containing the full-length HCV cDNA cloned into the cell which then infected with vTF7-3recombinant vaccinia helper viruses. This cell culture system is capable of producing high titer HCV RNA, and with the virus particles. Anyway, the recombinant HCV particles contain the pollution of the vaccin viruses, and it is inconvenience for the separation of virus particles. The vTF7-3recombinant vaccinia viruses is also toxic to cells, and in the cell culture system with vTF7-3as a helper virus, the cells will die soon. So we have to repeat the transfection and infection of vTF7-3and other procedures to culture the HCV particles, which is complicated and with some dangers of the experimental safety. In addition, these two cell culture systems need a helper plasmid or recombinant virus to provide trans-promoter activator and this is not the initial state of HCV RNA replication. Therefore, as a tool for study the HCV replication mechanisms and anti-HCV drug screening, it still has some limitations. Until now, there is no publication about the HCV genotype lb infectious cell culture has been established successfully in China.The goal of this study was to clarify whether the pretreatment genetic variations influence the outcome of antiviral therapy with PEG-IFN and ribavirin in Chinese patients with a subgenotype1b HCV infection. And on the basis of HCV1b replicon with adaptive mutation, to build the plasmids containing HCV1b full-length cDNA by replace the exogenous gene sequences in the1b replicon by Core-NS2gene frome the patients, and then establish the HCV1b full-length genome cell culture system. And hope it can play an important role in the research of the HCV in our country, For example, the study of the HCV life cycle, vaccine research, scanning for new anti-HCV compounds and so on.Chapter1Amplification of the5’terminal half genome and impact of its mutations of HCV genotype lb on the efficacy of the combined PEG-IFN/RBV therapy in Chinese patients with CHCMethodsThe serum samples of patients chronically infected with HCV genotype1b were selected. All of them have completed the combination therapy of180ug of pegylated IFN-a subcutaneously once weekly and oral ribavirin per day for48weeks; with6-months of follow up. Of the61patients enrolled in this study,35achieved a SVR at week72, while26were referred to as Non-SVR at that time. A single5.2kb fragment that spanned the region between and including the core to NS3genes was amplified. All PCR products were purified and consensus sequences for the5.2kb amplicons were obtained by direct sequencing and amino acid variations were compared.Statistical analysis:Statistical differences in the patients’ baseline parameters were determined by Student’s t-test for numerical variables and the chi-square probability test for categorical variables. The Mann-Whitney rank sum test was used to compare the number of all variations and unique variations between the SVR and the Non-SVR groups, while the proportions of unique variations and the association of these amino acid variations with treatment outcomes were compared using the chi-square test. The Mann-Whitney rank sum test was also used to compare the Shannon’s entropy values between the SVR and the Non-SVR groups. The average genetic distances between the groups were compared using an independent samples t-test. The level of significance for statistical significance was0.05. All statistical analyses were done using SPSS version13.0.ResultsIn this part of the study, we have shown that a SVR is associated with high levels of diversity in some regions of the pre-treatment, genotype1HCV genome. We compared the total number of variations and the number of unique variations relative to the consensus reference sequence for a genotype1b HCV in this population. The number and the proportion of unique variations(P=0.036, P<0.001and P=0.006, respectively; P<0.001, P<0.001and P<0.001, respectively), the total Shannon’s entropy values (P=0.019, P=0.023and P=0.046, respectively) and the mean genetic distances (P<0.001, P<0.001and P=0.011respectively) for the p7, NS2and NS3genes were all higher in the SVR than in the Non-SVR group. Thus, the primary conclusion of this study is that pretreatment genetic variations influence the outcome of antiviral therapy with PEG-IFN and ribavirin in Chinese patients with a subgenotype lb HCV infection.Chapter2Establishment of the HCV1b replicon cell culture systemsMethodsWe started with two plasmids gained as gifts and tried to establish the stable HCV lb replicon cell culture systems in Huh7.5.1cell. The fist one is lb-G replicon, which harbors the non-structure genomic cDNA of NS3-NS5B from HCV Conl isolate. And it has a adaptive mutation in NS5A (S2204I). The second one is1b-H replicon, which has a luciferase gene in the upstream of the neomycin phosphotransferase gene, compared with lb-G replicon. And it has three adaptive mutations in NS3(E1202G, T1280I) and NS4B (K1846T), respectively. We digested the b-H replicon and b-G replicon with Seal and then transcribed into RNA in phage T7RNA polymerase system in vitro. Transfected the RNA in Huh7.5.1cell, and followed by feeding with medium containing G418at800μg/ml. After several weeks, cell clones were isolated, proliferate and detected for HCV RNA by RT-PCR and HCV protein by indirect Immunofluorescent detection. Western Blot and luciferase assay.ResultsAs a result of this part,1b-H replicon and1b-G replicon conferred G418resisitance in Huh7.5.1cells. HCV RNA and protein were detected positively in the G418resistant cell clones. For1b-H replicon, we detected the expression of the luciferase reporter gene, instead of the time consuming RT-PCR and Western Blot, which greatly facilitates the experimental procedures.Chapter3Establishment of the full length HCV1b cell culture systemsMethodsA single5.2kb fragment of two patients, which were classified as’difficult to treat’chronic hepatitis C, was amplified and the PCR products were cloned.15-20clones were selected for quasispecies analysis by direct sequencing, and plasmids with the dominant sequence were introduced appropriate restriction enzyme sites by overlapping PCR. At last, the exogenous gene sequences in the lb replicon were replaced by the Core-NS2gene by restriction enzyme digestion in order to build the plasmids containing HCV lb full-length cDNA. We digested the plasmids containing HCV lb full-length cDNA with Seal and then transcribed into RNA in phage T7RNA polymerase system in vitro. Transfected the RNA in Huh7.5.1cell, and detected for HCV RNA in the cell supernatant and cell by RT-PCR and HCV protein by indirect Immunofluorescent detection, Western Blot, also the infectivity of the cell supernatant.ResultsOn the basis of1b-H replicon and lb-G replicon, we constructed4HCV full-length lb cDNA clones successfully by replacing the exogenous gene sequences in the1b replicon of the Core-NS2gene. After transfected the RNA into Huh7.5.1cells, we didn’t establish the infectious HCV cell culture system successfully, but the HCV RNA can replicate at a low level. When compared the1b-H-P2ã€1b-H-80and1b-G-P2ã€1b-G-80, which have a different adaptive mutations, we found that they replicated at a different HCV RNA level. This might suggest that adaptive mutations have a synergistic effect.ConclusionsOur results have shown that a SVR is associated with high levels of diversity in some regions of the pre-treatment, genotype1HCV genome, and the amino acids variations were focused in the p7, NS2and NS3proteins.The technique of amplification of a single5.2kb fragment laid foundation for establishment of the full length HCV lb cell culture systems. And the results of sequence analysis provided clues to the in vitro research about HCV protein and interferon resistance. Meanwhile, we have successfully established HCV1b replicon cell culture, which is the most prevalent subtype in China. This system will provide not only a new approach to the research of HCV pathogenesis and evaluation of antiviral drugs in our laboratory, but also a good platform to establish the genotype lb HCV full-length cell culture system.On the basis of1b-H replicon and1b-G replicon, we constructed4HCV full-length lb cDNA clones successfully. We didn’t establish the infectious HCV cell culture system successfully, but the HCV RNA can replicate at a low level. This might because there were some problem with the HCV assembly and release. Or it is maybe for only a little virus particles were secreted or the1b full-length HCV cDNA still have some defects, which can not produce complete recombinant virus particles. In order to exclude the latter reason, we used the infectious clone in the cell culture system with vTF7-3recombinant vaccinia helper viruses to construct the1b full-length HCV cDNA, and try to establish cell culture system, but still no infectious particles could be detected in the cell supernatant. This result suggested that there was no business with the sequence of lb HCV cDNA clones, which can not produce infectious virus particles. There are many factors will impact the virus replication, and more works should be done in further study to establish the lb HCV cell culture cystem. |
