Determination Of Dominant Variants Of Hepatitis C Virus Dynamic Quasispecies And Positive Selection Analysis Of Envelope 2 Gene Based On 6-kilobase Amplicons

Background and objective: HCV infection has been a global public health problem. It is estimated that about 210 million worldwide infected with HCV, in which 85% develop into chronic hepatitis C. However, existing anti-HCV treatment is very limited and no effective vaccines come out so far. An effective immune evasion of HCV, which mechanism is still not clear, leads to its persistent infection and has been the key problem in vaccine research. It is indicated that high variability of viral acclimatization rather than the defects or injury of host immune defense mechanism might play a more important role in viral immune evasion. HCV has obvious heterogeneity which presents a swarm of different but closely related variants(quasispecies) in the infected host. Furthermore, many studies show that there are two significant features in quasispecies. Firstly, variants is distributed unevenly with a strain existing as the dominant variants. Moreover, the dominant variants in individual is evolving continuously, which suggests the host immune pressure and strong adaptability of the virus. Thus, as the dynamic changes of dominant variants of quasispecies are related to HCV immune evasion, sequence analysis of long fragment of dynamic dominant variants would help explore the molecular basis of HCV immune evasion. Meanwhile, Establishment of the accurate identification of dynamic dominant variants would be an important prerequisite for study on quasispecis.HCV E2 gene exhibits a high degree of variability and is exposed to the strong selective environment of the host. The mutation under the strong host immune pressure might lead to corresponding changes for protein function, making the new variants escape the host immune clearance. However, the researches regarding the positive selected sites which non-synonymous nucleotide substitution rate(dN) exceeds its synonymous nucleotide substitution rate(dS) remarkably in HCV E2 gene are still limited.In this study, we got series of serum samples of genotype 1b chronic HCV patients without antiviral treatment screened from CQueen cohort. HCV 5' terminal 6Kb sequence was amplified by RT-PCR and cloned at efficiency. The method determining dominant variants of HCV dynamic quasispecies was established based on 6-kb amplicons. Simultaneously, we analyze the positive selected sites of E2 gene based on the 6Kb long fragment using fixed effects likelihood method(FEL) with consensus sequence of E2 gene as reference. Potential function affected by the involved sites were inferred.Materials and methods:1. Amplification of HCV 5' terminal 6Kb and its effective cloning. The series of serum samples were obtained from 3 cases of genotype 1b chronic HCV patients without antiviral treatment screened from CQueen cohort. The serum samples of 3 different time points studied were obtained at more than 6 months interval between 2 samples, with HCV RNA level of greater than 6log10 copies/ml. According to all the genome sequences of HCV 1b published in Genbank, the relatively conservative regions with appropriate GC content were selected for one RT primer and a pair of PCR primers. A clear 6Kb band was obtained using SuperScriptTMⅢRNaseH– reverse transcriptase and Platinum Taq DNA polymerase High Fidelity as well as the optimized RT and PCR conditions. The PCR products were cloned by Long fragment TOPO ? XL PCR Cloning Kit.2. Determination of dynamic dominant variants of quasispecies. 33 positive clones were selected randomly at each time point for 3 time potints from patient 1. There were 99 of sequences of 396 bp length spanning 5' terminal of E2 region to be analysed for determining the dominant variants change of serum samples at consecutive 3 time points by CLUSTAL_X, BioEdit and MEGA software. Every 10, 15, 20, 25, 30 clones were randomly selected from the 33 positive clones at the time point which quasispecies complexity is highest. Following repeating 100 times of process respectively to form 5 groups,χ2 test was used to accurately determine the least number of clones needed for detecting the dominant variants of quasispecies. Finally, the samples from another two patients were used to verify the results.3. Analysis of the positive selected sites of E2 gene. The positive selected sites of E2 gene based on the 6Kb long fragment were measured using FEL in HyPhy software(http://www.hyphy.org/) with consensus sequence of E2 gene as reference. Potential function affected by the involved sites were inferred compared with the known functional domain in E2 gene.Results:1. Establishment of amplification of HCV 5' terminal 6Kb fragment and effective cloning technique. The specific products was obtained from the serum samples by the optimal long RT-PCR with HCV RNA≥6log10 copies/ml. 200~800s recombinant clones can be acquired with positive recombinant ratio greater than 95% in one sample by using TOPO? XL PCR Cloning Kit.2. Establishment of the method determining dominant variants of HCV dynamic quasispecies based on 6Kb amplicons. The dominant variants of quasispecies of three time points in individul were located in differet clades in phylogenetic tree. The analysis of 5 groups of randomly picked clone sequences showed that the dominant variants of 25 and 30 clones is identical to that of 33 clones.3. Five positive selected sites were identified within the ectodomain of the E2 protein from 3 cases of genotype 1b chronic HCV patients. Meanwhile, it was found that quasispecies complexity in this region didn′t increase progressively as the infection time extended and however the quasispecies variants might reach homogeneity at some time point. These 5 sites identified in patient 1 and patient 3 located at HVR1, HVR2 and HCV E2-CD81 molecular binding regions: aa 384,aa 399,aa 410,aa 475 and aa 522.Conclusions:1. Amplification of HCV 5' terminal 6Kb fragment and effective cloning method were successfully established, which will provide a basic tool for the molecular study of HCV immune evasion.2. Twenty-five positive clones randomly picked up at each serum sample were sufficient to determine the dynamic dominant variants accurately during the quasispeies evolving. The dominant variants shift of HCV quasispecies may occur after more than six months apart.3. Several individual sites of HCV E2 gene based on 6Kb amplicon are under a strong host immune pressure, position aa 384, aa 399 and aa 410 may be involved in escape from neutralizing antibodies, while position aa 475,aa 522 may correlate to modulate the virus-receptor interaction which result in evading immunity. This study would lay a good foundation for analysis of positive selected sites in HCV C-NS4A gene.