| Part 1 Research on the Construction and characterization of HCV JFH1 Replicon with self-cleaving double ribozyme sequences in vitro and in vivoObjectivesHepatitis C virus(HCV) is a positive strand RNA viruses and the full-length genome is about 9.6kb which consists of coding and non coding region compose. The components of the coding region are core protein, envelope protein, p7 protein and non-structured region while the non coding is composed of 5`non coding region and 3`non coding region. HCV is the main pathogen causing chronic hepatitis C virus, cirrhosis and hepatoma. At present, more and more People have affected by the virus problem, but so far there is no corresponding effective vaccine. The WHO says there are the scopes of a total of 180 million patients with chronic hepatitis C virus. HCV has become the major public health problem in china even in the whole world.For now, the treatment of HCV mainly depends on the pegylated interferon plus ribavirin. Due to the limited efficacy and the curative effects are significant differences between different HCV genotype. It is urgent immediate to develop more effective anti HCV drugs to provide help for the clinical treatment. But the main obstacle to the development of new anti HCV drug is lack of convenient model for HCV infection. At present, there are some researches base on JFH1 multiple polycistronic, monocistronic HCV genomic and subgenomic replicon, but this kind of replicon can only start the replication of the virus, can not generate infectious viral particles in cells, limiting the research on biological characteristics of HCV. The whole JFH1 genome RNA transits by Kato through artificial transcription, electric transfected into Huh7.5 cells to start the synthesis of HCV.But this method of operation is very complex, and can not be used for animal experiment; neither can build a stable transfected cell line, considerable limitations. In the vitro model, chimpanzee is sensitive to HCV shows a similar course of chronic hepatitis, but because of the economic and ethical issues, it is difficult to chimpanzee used as animal model. In view of the above reasons, for the establishment of the HCV genome in the vitro and viral replication model, is an important foundation for further research work of HCV. We establish a full-genomic HCV replicon with self-cleaving double ribozyme sequences according to study the JFH1 sequence of c DNA. The replicon can not only start the replication of HCV directly, but also generate infectious viral particles in cells. The virus genome through replication, packaging, release and reinfection to maintain the long and efficient replication of HCV training, simulating the HCV complete life cycle. This replicon can be used to observe and study on the biological characteristics of HCV and probe into and screen HCV drug over a long period of time. Studies on the replication can also be carried out in the animal model.Methods1. Self-cleaving ribozyme sequences were added to each side of HCV JFH1 and JFH1/GND c DNA, then inserted these DNA into the downstream of CMV promoter of pc DNA3.1 expression vector to construct the recombinant plasmids which named pc DNA3.1-RZ-JFH1 and pc DNA3.1-RZ-JFH1/GND.2. Those recombinant plasmids were transfected into Huh7.5 cells(5μg/100 mm culture dish) and injected into Kunming mice by high-pressure tail vein injection(10~30μg/mouse). immunofluorescence、immunohistochemistry 、q RT-PCR and serological testing were used to testify the replication properties of recombinant plasmids in vitro and in vivo.Results1. Construction of full-genomic HCV JFH1 replicon with b self-cleaving double ribozyme sequences : the recombinant plasmid of pc DNA3.1-RZ-JFH1 and pc DNA3.1-RZ-JFH1/GND which were appraised by Eco RI and Xba I. Those enzyme bands were consistent with the expected size. The recombinant plasmids were successfully constructed.2. During the 16 week’s test, 1×106 ~ 3×106 copies /ml HCV RNA was detected from the supernatant which the replicon pc DNA3.1-RZ-JFH1 transfected Huh7.5 cells. The culture supernatant of pc DNA3.1-RZ-JFH1/GND transfected cells in the HCV RNA level was below the detection limit.3. The replicon pc DNA3.1-RZ-JFH1 transfection group cells of HCV antigen positive cells were about 20%; The infection group of HCV antigen positive cells were about 5%. The viral particles which produced by the replicon could infect na?ve Huh7.5 cells.4. In high-pressure tail vein injected Kunming mice, only transient viremia was observed without finding any HCV antigen positive cells by immunohistochemistry and observing any HCV specific antibodis by serological testings.ConclusionConstruction of HCV JFH1 replicon can generate infectious viral particles in vitro long-term, steadily and efficiently while failed to obtain the expected effect in vivo experiment. It may concern with the specificity of HCV JFH1 strain. The HCV JFH1 replicon can be used to study the HCV virus and antiviral drug screening etc.Part 2 Research on the clinical effect of pegylated interferon plus ribavirin for HCV genotype 6ObjectivesAt present, pegylated interferon plus ribavirin is the standard antivirus solutions for HCV infection in the clinical treatment, but the effect is depends on the genotype of HCV infection. There are obvious differences in different genotypes of sustained virological response rates. It has been suggested that HCV genotype 1 was lowest, only 40- 50 %, HCV 2/3 can reach 70-80%, or even higher. HCV has a total of 6 genotypes. HCV genotype 1, 2, 3 are widely distributed globally. HCV genotype 4/5 are found in the Africa while HCV genotype 6 is prevalent in Asia. As the world economy and medical pattern, few studies have been carried out in HCV 4, 5, 6. The main researches focus on HCV genotype 1, 2 and 3. In recent years, with medical and health conditions improved in China, HCV changed the mode of transmission. HCV infection due to blood transfusion and use blood products have decreased significantly,however,the number of intravenous drug user has increased. That people who is intravenous drug users become the main cause of HCV genotype 6 infection in our country, so the HCV genotype 6 infection rate is on the rise. Studies have shown that this trend is particularly in the southwest region of China. Maybe this is because that southwest region of China is adjacent to Southeast Asia, which is one of the world’s most important opium planting and drug producing bases.Owing to the rate of HCV infection has been increasing speedily, we must standard our clinical treatments. The infection has the standardized treatment in HCV genotype 1, 2 and 3. But HCV genotype 6 infection treatment and methods still need further exploration, and to determine the optimal. In recent years, studies have compared the effect of antiviral treatment of HCV genotype 1、2、3 and genotype 6. They thought that the treatment response rate of HCV genotype 6 infection is higher than HCV genotype 1, and treatment response rate of HCV genotype 2/3 infection had no significant difference. There are also some comparative studies of therapeutic effect of HCV genotype 6 antiviral treatment for 24 weeks and 48 weeks, but it is no statistically significant differences between the two effects. Now comes a report that pegylated interferon combined with ribavirin treatment of 24 W for hepatitis C genotype 6 infection have a similar effect with HCV genotype 2/3. We collected all clinical data of infectious diseases of Chongqing Southwest Hospital from May 2010 to October 2014 which applied PEG-IFN plus RBV in treatment of HCV genotype 6 infection and HCV genotype 2/3 infection. In the analysis, we ultimately determine 53 cases in group HCV genotype 6 infection and 84 cases HCV genotype 2/3 infection through rigorous process of selection. By comparing the therapeutic response of the two groups of curative effect, guides the clinical treatment.MethodsWe collected all clinical data( Age, gender, serum HCV RNA,s ALT,color Doppler ultrasound results, the treatment process, the route of infection)of infectious diseases of Chongqing Southwest Hospital from May 2010 to October 2014 which applied PEG-IFN plus RBV in treatment of HCV genotype 6 infection and HCV genotype 2/3 infection.All of the patients with HCV genotype 6 and 2/3 infection were meet the following criteria:(1)Patients who were treated with either PEG-IFNa-2a or PEG-IFN a-2b plus RBV. RBV was given at a daily dose of 800 mg, 1000 mg, 1200 mg for those weighing <65kg, 65–85kg,>85 kg,respectively. PEG-IFN α-2a was given 180μg/week subcutaneously, and PEG-IFNα-2b was given at a weekly dose of 1.5 μg/kg.(2)The time of their treatment is not only more than 24 weeks but also less than 26 weeks, and follow-up time reached more than 24 weeks.(3) The exclusion of HBV, Autoimmune Liver Disease,HIV, Hypertension, Diabetes.HCV genotype 6 as experimental group,HCV genotype 2/3 as control group. Respectively on two groups of patients with RVR, EVR, SVR comparison.All statistical analyses were performed with SPSS 18.0. Measurement data were expressed as x±s. Comparison of baseline measurement data using student’s-test. Categorical variables were analysed using the Pearson chi-squared test or Fisher ’s exact test as appropriate. Multivariate analysis were performed using Multiple logistic regression analysis.The efficacy of PEG-IFN plus RBV was evaluated by the non-inferiority test, When the efficacy of the experimental groups is not less effective than the control group, the non-inferiority test is effective.With the help of clinical professor,we set the non-inferiority margin to 15%. Differences with a two-tailed P-value≤0.05 were considered statistically significant.Results1. The ages, gender and the serum HCV RNA, s ALT level and the variety of applied PEG-IFN in pre-treatment of HCV genotype 6 infected and HCV genotype 2/3 infected has no statistically significant difference. But in the history of intravenous drug users, the proportion of HCV genotype 6 infected is higher than HCV genotype 2/3( P=0.001).2. The ratio of patients infected with HCV genotype 6 and 2/3 got RVR is 88.7% and 89.3% respectively,95% confidence interval for the rate difference is:(-0.1139,0.1019);EVR is 94.3% and 96.4%,95% confidence interval for the rate difference is:(-0.0950,0.0530);SVR is 86.8% and 90.5%,95% confidence interval for the rate difference is:(-0.1476,0.0736);the positive predictive value of RVR to SVR is 89.4% and 93.3%,95% confidence interval for the rate difference is:(-0.1436,0.0656); EVR to SVR is 88.0% and 91.4%,95% confidence interval for the rate difference is:(-0.1428,0.0748).The floor level of 95% confidence interval for the rate difference of the above indexes are less than non-inferiority margin(0.15),so the non-inferiority test is true. From the comparison of the above indexes, we can draw a conclusion that HCV genotype 6 infected is not less effective than HCV genotype 2/3 infected.3. whether the HCV 6 infected are drug abusers, each of the influence of treatment response has no statistically significant differences.ConclusionWith Pegylated interferon plus ribavirin, the curative effect of HCV genotype 6 infected is not less effective than HCV genotype 2/3 infected. Therefore, we can use the twenty-four week’s PEG-IFN plus RBV as the standard therapeutic schedule of clinical HCV genotype 6 infected. |
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