Establishment Of Chinese Dicistronic Subgenomic Replicon Of Hepatitis C Virus

Background and objective: Hepatitis C virus is the cause of the hepatitis C, which is a global health problem. The World Health Organization estimates that 170 million people, 3% of the world population, are infected with hepatitis C virus (HCV). The majority of those infected (55–85%) fail to clear the virus and become chronic carriers manifested by the persistent presence of detectable virus in the serum. About 20% -50 % of patients with chronic hepatitis C will develop liver cirrhosis within 20 years. Once cirrhosis is established, the rate of hepatocellular cancer development is 1-4% per year. The standard treatment for chronic HCV infection is pegylated -interferon in combination with the ribavirin. About 54 %-56 % of patients respond to the therapy and show a sustained reduction in viral titer. However, many patients do not qualify for or do not tolerate standard therapy. Therefore, more effective and better tolerated therapeutic strategies are urgently needed. But their development has been significantly delayed by the lack of appropriate cell culture models or small animal model permissive for HCV that allow the propagation of the virus in the laboratory. Recently, the development of selectable dicistronic subgenomic replicons of HCV has provided a new research tool for hepatitis C. These in vitro systems can support efficient HCV RNA replication and synthesis of viral proteins.HCV is a positive, single-stranded RNA virus in the Flaviviridae family. HCV is divided into six major genotypes, within each genotype more closely related isolates are grouped into different subtypes. HCV genotypes and subtypes are distributed differently in different parts of the world, and certain genotypes predominate in certain areas.Genotypes 1-3 are widely distributed throughout the world. Subtype 1a is prevalent in North and South America, Europe, and Australia. Subtype 1b is common in North America and Europe, and is also found in parts of Asia. Genotype 2 is present in most developed countries, but is less common than genotype 1. Therefore,it will be very important to get the prevalent HCV Subtype by investigating the distribution status of hepatitis C virus genotypes in China, which will be useful for further construction of Chinese replicon of subgenomic hepatitis C virus. HCV RNA genome is approximately 9.6 kb in length with complicated secondary structure and plenty of G or C base in long viral RNA templates which make long reverse transcriptase polymerase chain reaction (RT-PCR) of HCV difficult. And previous studies have amplified up to 20 fragments, each less than 1 kb in length or used a mixture of PCR fragments and cloned cDNA libraries prepared from clinical specimens to get the entire HCV genome. But the HCV contains so many quasispeices. It is obvious that the entire HCV genome which constructed by short RT-PCR fragments splicing may not provid a reliable tool for the study of HCV mutation, infectious clone and vaccine. HCV 3' UTR comprised the variable region (VR) located at the 5' end of the 3'UTR, an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X-tail).Each of these domains was found to contribute to efficient replication of the viral RNA. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. The longer fragments of the HCV genome would allow more sequence information to be obtained and provide more detailed information about HCV.In the study, we established Chinese HCV and determined genotypes and subtypes of HCV RNA positive serum specimens. Genotype 1b is the prevalent strain in China repository and. Thus, Chinese HCV genotype 1b replicon systerms were established in the study, which will provide a useful tool for the study on the HCV pathogenesis, the screening of antiviral drugs and the development of vaccine.Materials and methods:1. The establishment of HCV repository: serum specimens of HCV infected in- patients and out-patients from Infections Diseases Department were continually collected since 2004. All specimens were conducted for routine biochemistry and virology test. We applied special software to manage the related data of specimens, including epidemiology information, clinical and laboratory data. Simultaneously, genotypes and subtypes of a part of HCV RNA positive serum specimens were determined by nucleotide sequencing and phylogenetic analysis. RT- PCR primers for genotyping were deduced from the relatively conserved regions: core-envelope 1 (C-E1) region. Amplicon fragment is 474nt in length.2. The establishment and optimization of the long RT-PCR of HCV hemigenome: 5'and 3'terminal half of the HCV-1b genome were respectively amplified using optimized primers. Total RNA was extracted by using 3 different methods including TRIzol LS Reagent, QIAamp viral RNA mini kit and Magnetic bead. The extracted RNA was respectively reversely transcribed with 3 different reverse transcriptases under different reaction conditions. Optimization for long PCR amplification was performed by comparing different Taq enzymes and cycling systems.3. The construction of Chinese genotype 1b replicon: we selected the HCV subtype 1b serum specimen with high viral load as the template for amplification of long HCV fragments by the the refined long RT-PCR technique. Full-length HCV genome plasmid was obtained by two long fragment splicing and TA cloning. HCV NS3-3'UTR gene fragment can be amplified using the full-length plasmid as templete, then, replace the HCV nonstructural region of the plasmid pNNeo/3-5B to establish the Chinese replicon plasmid. Plasmid DNA was extracted and linearized to form transcript-RNA using T7 RNA in vitro transcription system. Huh-7 cells were transfected using Lipofectamine 2000 packaging transcript-RNA, followed by selection with G418 to get resistant clones. The target replicon was identified by RT-PCR,indirect immunofluorescence and western blot for efficient RNA replication and the expression of HCV proteins.Results:1. Establishment of HCV repository with success: up to now, we have collected 934 serum specimens from 605 HCV infected patients, and recorded epidemiology information, clinical and laboratory test data of all patients in a detail. Genotypes and subtypes of 206 HCV RNA positive serum specimens were determined, which contains five different subtypes of HCV: 1b(42.2%), 2a(12.6%), 3a(15.5%), 3b(14.6%), 6a(15.1%). Two subtypes, 1a and 2b, were not found. 1b is the prevalent genotype.2. Establishment of long RT-PCR of hemigenomic HCV: A refined Long RT-PCR amplification of HCV hemigenomic fragment was established by using SuperScriptTMⅡRNase H–reverse transcriptase and Platinum Taq DNA polymerase High Fidelity as well as the suitable cycling conditions. The amplification of HCV 5'hemigenome of 6 serum specimens can get specific products by the optimal long RT-PCR technique. The amplification of 3'terminal hemigenomic fragment have gained specific products, but the Sensitivity, repeatability and specificity need to be further improved.3. Construction of Chinese genotype 1b replicon: Full length HCV genome plasmid was constructed and used as templete to get Chinese 1b NS3-3'UTR. The Chinese 1b subgenomic replicon plasmid was obtained by using Chinese 1b NS3-3'UTR to replace the counterpart of pNNeo/3-5B. G418-resistant replicon clones were formed by selection of transcript-RNA transfected Huh-7 cells after about 45 days. RT-PCR confirmed that the replicon RNA efficiently replicated in clone cells. The expression of HCV NS5A and NS3 proteins was validated by indirect immunofluorescence and Western blot.Conclusions:1. HCV repository was established, which will provide a basis for the HCV basic and clinical study.2. Chinese HCV 1b subgenomic replicon was established. It is a useful tool for the study on HCV pathogenesis, the screening of antiviral drugs and the development of vaccine.