Amplification And Sequence Analysis Of HCV 1b Full-length Genome

Background and objective:Hepatitis C virus (HCV) infection has been a global public health problem. It is estimated that up to 170 million people world wide are infected with HCV, 85% of them eventually develop into chronic hepatitis C. There is no vaccine to prevent HCV infection so far, the only one standard treatment for chronic HCV infection is combination treatment with pegylated-interferon and ribavirin, but the curative effect on China's popular 1b strains is limited. The lack of an effective cell culture system and small animal model for HCV has slowed HCV pathogenic mechanism research and antiviral drug development. The HCV subgenomic replicon constructed by Lohmann et al. provided a new platform for HCV molecular studies and antiviral drugs screening. However, since the complete structural region was deleted, virus assembly and particle release could not be studied with this system. Moreover, important information about a potential influence of the structural proteins on the host cells could not be obtained. The establishment of JFH1-based cell culture model is a milestone of HCV research. JFH-1 was isolated from a Japanese fulminant hepatitis patient, which represented the only cloned HCV wild-type sequence capable of efficient replication in cell culture without adaptive mutation. HCV displays high genetic heterogeneity. Based on sequence diversity, HCV strains are divided into six major genotypes, within each genotype, closely related variants are grouped into subtypes. JFH-1 strain belongs to HCV genotype 2a, it can not represent the characteristics of all HCV strains due to the great differences in biological and clinical characteristics between different HCV genotypes. It is more urgent to establish cell culture systems of all HCV genotypes especially 1b subtype which is the most common HCV variant in China.Construction of full-length HCV genome plasmid is the basis for establishing HCV cell culture system. The main method to obtain full-length HCV genome is to splice overlapped RT-PCR products. However, the full-length HCV genome plasmid constructed based on multiple fragments splicing is artificial in concept and is not necessarily the dominant viral variant. Therefore, full-length HCV genome obtained through long RT-nested-PCR is more reliable and useful. HCV RNA genome is approximately 9.6kb with complicated secondary structure along the whole genome. Moreover, HCV can not be cultured in vitro, the only source of RNA template for long RT-nested-PCR is clinical samples in which HCV has a low titer. At present, the longest fragment of HCV genome obtained through long RT-nested-PCR is 9.1kb. It can not be used for the construction of HCV cell culture system because the lack of 3'UTR, which has been confirmed as an indispensable element in the replication of HCV.In this study, we established a refined long RT-nested-PCR for amplifying full-length HCV genome from patient serum and constructed six full-length HCV genome plasmids, which will provide a platform for the construction of HCV cell culture system.Materials and methods:1. The establishment and optimization of long PCR for amplifying HCV full-length genome:Optimization of long PCR strategies by testing a series of primers, adding various concentrations of glycerol or DMSO, or both, using different cycle systems, then selected the optimal long PCR conditions.2.The establishment and optimization of long RT- nested-PCR for amplifying HCV full-length genome:Optimization of long RT-nested-PCR strategies by testing different RT and PCR primers, improving RNA extraction method, optimizing long RT system, verifying the integrity of cDNA, altering concentration of long PCR primers, then choose the optimal long RT-nested-PCR conditions.3.The construction of full-length HCV genome plasmid:The full-length HCV genome fragment was inserted into pCR-XL-TOPO vector and transformed into TOP10 competent cells.4.The analysis of HCV full-length sequences:Six plasmids were sequenced by Sequencer ABI 310. Complete sequences were obtained by software Vector NTI Suite 8. Phylogenetic tree was drawn to understand the origin of cloned HCV strains.Results:1.Establishment of long RT-nested-PCR for the efficient amplification of full-length HCV genome:we have investigated each step of the long RT-nested-PCR procedure and developed a refined protocol for the amplification of full-length HCV genome from serum by adding 10% glycerol into long RT and PCR system, using SuperScriptⅢreverse transcriptase and Platinum Taq DNA Polymerase High Fidelity as well as the suitable cycling conditions.2.Construction of HCV full length genome plasmid:Six full-length HCV genome plasmids were achieved by TA cloning(CQFL1~6) and used as template to amplify HCV full-length fragment successfully.3.The phylogenetic tree indicated that cloned HCV strain was HCV genotype 1b. NS5B amino acid sequence analysis between CQFL1~6 and other HCV 1b strains discovered that 4 amino acids exclusively in CQFL1~6 from other 1b strains, three of them were the same with the corresponding amino acid in JFH-1. We speculated that the spatial structure of CQFL1~6 maybe more close to JFH-1 than other HCV 1b strains and may have the ability to replicate in vitro.Conclusions:1 . Long RT-nested-PCR method for amplifying full-length HCV genome was established. The technology described in our study should be applicable to other HCV genotypes as well as other RNA viruses.2.Full-length HCV genome plasmids were successfully constructed. Our work could support the development of selectable full-length genome replicon which is a useful tool for HCV researches.