| BackgroundHepatitis C virus (HCV) is a major etiological agent of chronic hepatitis, and among these,20% to 30% will develop into liver cirrhosis and hepatocellular carcinoma (HCC) within 10 to 30 years of infection. It has been estimated that there are 180 million patients infected with HCV worldwide. The high persistence of HCV infections, together with the low susceptibility to antiviral treatments, is probably due to a complex interaction between the genetic diversity of the virus and the host immune response. So far, HCV infection is still one of the severely public health problems in world. HCV is belonging to Flaviviridae, and a member of hepatitis virus. Its genome is a single strand RNA, with whole genome about 9.6kb. Since lack of proofreading capability of the viral RNA polymerase, the RNA genome of HCV demonstrates profound heterogeneity at the sequence level. HCV genotypes are determined based on the divergence of genome nucleotide sequence between 30% and 35% and the subtypes on the divergence of genome nucleotide sequence between 20% and 25%. To date, HCV is classified into six genotypes and more than 80 subtypes.HCV genotype distribution has special geographical and ethnic characteristics. However, the distribution of HCV genotype presents a fixed epidemic model. With the global population migration and mobility, as well as the shift of the mode of transmission, HCV genotype distribution also changes. Studies have shown that HCV genotype distribution is changing in some countries, such as France, Italy, Poland, Germany and other countries. With the rapid development of China’s economy, as well as increasing exchanges in many fields between different regions, it leads to population mobility and provides the environment of HCV spread. Due to The implementation of HCV antibody screening in 1993, the route of transmission of HCV has change from the original transfusion transmission to the coexist of the route of blood transfusions, drug use, tattoo, sexual transmission and so on. The massive movement of population as well as the change of the transmission mode, HCV genotype distribution might be changing. Previous studies have shown that in China, subtype lb is the prevalent subtype, followed by subtype 2a, and the proportion of patients with subtype 2a in northern areas is higher than that in the south. Studies showed that the prevalence of lb subtypes was about 90%, subtype 2a around 8%, and subtype 6a was not detected from 1997 to 2002 in southern China. In 2005, a study, enrolled the small number of HCV RNA positive samples, showed that genotype 6a replaced 2a as the second common genotype in the Pearl River Delta, Guangdong province. However, the conclusions need to be further confirmed because of the small sample size.The viral factors influencing the outcome of antiviral therapy of chronic hepatitis C includes HCV genotype, the mutation of Core and NS5A regions, the pretreatment viral load etc. Studies have demonstrated that HCV genotype is associated with the efficacy of antiviral therapy and the course of treatment. "The prevention and treatment guidelines of HCV infection" published in China in 2004 as well as "the clinical practice guidelines of HCV infection (update)" revised by AASLD in 2009 have been made some suggestions:patients infected with genotype 2/3 respond better than those with genotype 1; in addition, patients infected with genotype 1 should be treated for 48 weeks with peginterferon plus ribavirin, whereas patients with genotype 2/3 could be treated with SOC for 24 weeks. Thus, HCV genotype has the important clinical significance in guiding patients for antiviral therapy. Genotype 6 is rare and confined to some regions. There are very few data available on its outcome of antiviral therapy and the factors. In view of the rapid transmission of subtype 6a in southern China, the study of its clinical features, the outcome of antiviral therapy and predictors for treatment in comparsion with subtype 1b is important for clinical practices.The combination of pegylated interferon (pegIFN) and ribavirin (RBV) is the current standard of care (SOC) therapy for chronic hepatitis C, but only approximately 50% of patients can achieve sustained virological response. In light of side effects and high costs of SOC, it is important to identify factors for predicting an individual’s response before treatment. Therefore, it is important for us to explore the the predictors of treatment response for carrying out the individual therapy and avoiding side effects. Recently, genome-wide association studies (GWASs) showed that single nucleotide polymorphism (SNP) (rs 12979860 or rs8099917) near the gene IL28B coding for IFNλ3 was associated with the antiviral treatment response of the combination therapy of pegIFN plus RBV. The discovery indicates that the study of HCV enters a new era. The establishment of IL28B genotyping assay which is rapid, simple and reliable is one of research hotspots.For a long time, little progress has been made on the study of HCV cell culture model. In 2005, Wakita et al. setup the infectious HCV genotype 2a cell culture system successfully. The robust HCV cell culture infection system based on the HCV JFH-1 molecular clone allows the production of virus that can be efficiently propagated in tissue culture. The construction of JFH1 cell culture was considered as the breakthrough in the history development of cell culture technology. The cell culture model of subtype 2a did not meet the research needs due to the differences of biological characteristics and response to antiviral therapy between HCV genotypes. Subsequently, many scholars tried to construct the HCV whole genome cell culture system of other subtypes. However, little progress has been made. Recently, the chimeric genotype cell culture system has been made some progress on basis of JFH1 cell culture model. To date, some chimeric virus cell culture systems, including 1a/2a, lb/2a,3a/2a,4a/2a,5a/2a,6a/2a and 7a/2a, has been successfully constructed. In view of the rapid growth of subtype 6a in southern China and the spread to the whole country, the biological characteristics, vaccine development, evaluation and screening of antiviral agent have become urgent needs of subtype 6a research. Therefore, it is necessary to establish subtype 6a cell culture system. So far, HCV 6a subtype cell culture system has not been successfully constructed.AimsBased on the preliminary results of HCV genotyping research from our team, we intend to enlarge the sample size to investigate the HCV subtype distribution and its characteristics among CHC patients in southern China, to analyze the shift of HCV subtype, to explore the reason of changes of HCV subtype distribution and the source of subtype 6a.To analyze the clinical baseline characteristics of CHC patients infected with subtype 6a in comparison with those with subtype 1b, and to explore the outcome of antiviral treatment and the impact factors of viral response to therapy. To develop an assay, allowing the identification of IL28B rs8099917 genotype related to the outcome of antiviral therapy, and to analyze the association of rs8099917 genotype with the treatment outcome.In view of the rapid growth of subtype 6a in southern China and the spread to the whole country, we intend to construct 6a/JFH1 recombinant viral plasmid for providing the experiment basis of the development of subtype 6a genome cell culture system, with the aim of study on biological characteristics, vaccine development, evaluation and screening of antiviral agent of subtype 6a.Methods1. The distribution of HCV genotypes and subtypes among CHC patients: Genotypes or subtypes were determined by the phylogenetic analysis of directly sequenced C/El and NS5B regions by optimizing the primers. Patients’data, including gender, age, HCV RNA load, routes of transmission and duration of HCV infection, were collected from clinical records. Logistic regression model was used to analyze the risk factors associated with HCV genotype. We analyzed the shift of HCV subtype and explored the reason of changes of HCV subtype distribution and the source of subtype 6a.2. The study of viral and host factors of influencing on the outcome of antiviral therapy:the establishment of clinical research in patients infected with subtype 6a compared with those with subtype lb. All of them completed the combination of pegylated interferon and ribavirin for 48 weeks, with 6-months of follow-up. The clinical data, including gender, age, HCV RNA load, ALT, AST, etc. at pretreatment baseline, as well as HCV RNA load at weeks 4,12,48 of treatment and 24 weeks after the therapy were collected. Statistical analysis was used to observe the clinical baseline characteristics of CHC patients infected with subtype 6a in comparison with those with subtype lb, and to explore the outcome of antiviral treatment and the impact factors of viral response to therapy. 3. The establishment and application of the assay for IL28B rs8099917 genotyping:We established the use of tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) for detecting IL28B rs8099917 genotype. The assay specificity was confirmed by direct sequencing and positive control. We used direct sequencing to detect rs 12979860 genotypes.4. The construction of 6a/JFH1 recombinant viral plasmid:the Core-NS2 gene of HCV 6a and JFH1 partial gene fragment were connected by the Overlapping PCR. 6a/JFH1 recombinant virus was constructed via endonuclease digestion.5. Statistical analysis:The chi-square probability test was used for categorical variables. Student’s t-test or Mann-Whitney rank sum test was applied to compare measurement data between two groups, and One-Way ANOVA for those among three groups. Multinomial logistic regression model was established to analyse factors associated with HCV genotypes. Binary logistic regression model was used to explore predictors for SVR in CHC patients. The level of significance for statistical significance was 0.05. All statistical analyses were done using SPSS version 13.0.Results1. Among the 388 samples, there were overall eight genotypes detected. The most prevalent genotype was lb (59.3%,230/388); the next common genotype was 6a (21.6%,84/388), followed by 2a (8.0%,31/388),3a (5.2%,30/388),3b (4.1%, 16/388), la (1.3%,5/388),2b (0.3%,1/388) and 6d (0.3%,1/388). Genotype 6d was firstly detected in Southern China. Genotype 6d was firstly detected in Southern China.2. HCV genotype distribution changed significantly according to the age. Patients infected with genotype 6a were younger than those infected genotype lb or 2/3, with the mean age 34.9±8.6 years compared with 42.7±15.3 and 40.2±10.7 years, respectively (P<0.001). Patients infected with genotype lb presented to the increasing tendency with age. However, individuals infected with genotype 6a were found to decrease consecutively with age except ≤25 years group.3. The gender distribution was found to vary for different genotypes (p=0.011). Males accounted for higher percentage than females in patients infected with genotype 6a or 2/3 (76.2% vs.23.8% and 67.6% vs.32.4%, respectively).4. The distribution of HCV genotypes showed significant differences depending on the route of transmission (P<0.001). Genotype 1b was observed more frequently (80.5%) in patients infected through transfusion than other routes, while genotype 6a were more frequent (63.3%) in those infected through IDU. There were notable variations in the distribution of 117 individuals infected with genotype lb according to the year at infection. HCV acquisition before 1995 accounted for the predominant proportion of 84.6%(99/117) in patients infected through transfusion, and after 1995, it dropped dramatically and subsequently remained stably at a lower lever.5. In multivariate logistic regression analysis, only transmission routes were independently associated with HCV genotype. Genotype lb was significantly more common in patients infected through transfusion (P<0.001), while genotype 6a was observed more frequently in patients infected IDU (P<0.001).6. Phylogenetic analysis based on the C/E1 and NS5B regions showed that genotype 6a of Southern China presented a high degree of similarity to that of Hongkong. Both of them were close to genotype 6a strains of Vietnam in term of evolutionary distance.7.47 CHC patients infected with genotype lb and 41 cases with genotype 6 were enrolled in this study. There were no significant differences in gender, age, ALT, AST, ALB, TBIL, TG, CHOL, GLU, WBC, RBC, HGB, PLT and HCV RNA load at baseline characteristics (P<0.05).8. Among 88 CHC patients,63 achieved an SVR (71.6%). Compared with patients infected with 1b, those infected with 6a had the higher rate of RVR and SVR (92.7% vs.66%, P=0.002 and 85.4% vs.59.6%, P=0.007, respectively). However, there was a similar rate of EVR and ETVR (95.1% vs.87.2%, P=0.362 and 95.1% vs.91.5%, P=0.802, respectively).9. In multivariate logistic regression analysis, HCV genotype (0R=3.59, P=0.036) and age (0R=0.92, P=0.001) were independently associated with SVR before antiviral therapy, while RVR (0R=17.24, P=0.012) and age (0R=0.88, P=0.001) were independently associated with SVR after the start of treatment.10. We developed a rapid, simple and reliable assay, allowing the identification of IL28B rs8099917 genotyping to predict the response to IFN-based treatment. The genotyping results of the T-ARMS-PCR were confirmed by direct Sanger sequencing of amplified PCR products using the outer primers in all samples, and showed correspondence with that by DNA sequencing in 55 samples (98.2%).11. Among 56 patients with chronic HCV-1b infection, for IL28B rs8099917 genotyping, TT genotype and TG genotype accounted for 80.4%(45/56) and 19.6%(11/56), respectively. GG genotype was not found. Patients with TT genotype could chieve better virrological response than those with TG genotype (68.9% vs.27.3%, p=0.029). We used direct sequencing to detect rs12979860 genotypes, and also found the strong linkage disequilibrium between the two SNPs in Chinese population.12. We successfully constructed the 6a/JFH1 recombinant viral plasmid. However, after transfection into Huh7.5.1 cell, the plasmid showed lower replication activities and needed further development for screen adapted cell lines.Conclusions1. In Southern China, among chronic hepatitis C patients, there were eight subtypes, including 1a,1b,2a,2b,3a,3b,6a and 6d. The most prevalent subtype was 1b, and the next common genotype was 6a. The study provided a new picture of HCV genotype distribution in Southern China, with a decreasing prevalence of genotype lb associated with transfusion infection route and an increasing frequency of genotype 6a associated with intravenous drug use (IDU).2. Patient baseline clinical characteristics were similar between HCV subtype 6a and subtype lb. But the outcome of antiviral therapy was different. Compared with patients infected with subtype 1b, those infected with subtype 6a could achieve a higher rate of SVR. HCV genotype was the important predictor for SVR before treatment. But, after 4 weeks of therapy, RVR was the crucial predictor for SVR.3. T-ARMS-PCR was a sample, effective and economical assay, allowing the identification of IL28B rs8099917 genotype that was associated with the outcome of antiviral therapy. There was the strong association between rs8099917 and rs12979860 in Chinese patients.4. In this study, the 6a/JFH1 recombinant viral plasmid was successfully constructed to prepare for the experimental basis of study on subtype 6a cell culture system of the whole genome in future. |
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