| AimHepatitis C virus exist in highly related variants called quasispecies in individuals. This character is very important for HCV infection to predispose chro-nicity and resistant to IFN therapy. In order to explore the relationship between HCV quasispecies and the effectiveness of IFN therapy, we detected the quasispecies in the region of HCV HVR1 and NS5A using different methods in our present study. This study has great clinic significance for the selection of the patients received IFN therapy and the prediction of the thereputic results before the treatment of IFN.Materials and methods1. Patients; Twenty patients infected with chronic hepatitis C who were treated with IFN - a were studied. All of the patients received interferon alfa for six months in a dose of 3 million units intramuscularly three times a week and have been followed for six months at least after therapy. End of treatment responses were definded as the normalization of alanine aminotransferase and negative HCV RNA when the treatment was over; Complete responses were definded as the persistent normalization of alanine aminotransferase and negative HCV RNA for 6 months at least after therapy; Others were definded as nonresponders. Sera of the patients before treatment were collected and stored at - 70℃ until use.2. The genome of NS5A and HVR1 region were amplified by the methods of Reverse Transcription Polymerase Chain Reaction ( RT - PCR). The primers were designed according to the conserve parts of two sides of HVR1 and NS5A region respectively.3. HCV genotypes were classified using the methods of genotype specifiedPCR and direct sequencing. In the method of genotype specified PCR, one upstream primer and four different downstream primer were used. The necleotide length of I , II , III and IV genotypes were 57bp, 144bp, 174bp and 123bp respectively. Meanwhile ,some cases were classified by direct sequencing acceding to the necleotide sequences of NS5A region.4. The HVR1 quasispecies heterogeneity was detected by cloning and sequencing, the PCR products having been purified were ligated with pDrive Cloning Vector, then transformed into QIAGEN EZ Competent Cells, the white re-cornbinant clonies were selected from LB agra plates ( 10 clonies of each samples were selected). After the recombinant clonies were abstracted and confirmed by PCR,the detection of necleotide sequences of HVR1 region were performed.5. HCV RNA level was detected using the method of specified fluorogence probe PCR. This method combined the technique of PCR amplification with fluorogence probe, through detecting the change of the intensity of fluorogence in PCR reaction system,HCV RNA quantity could be presumed.Results1. Genome of HVR1 and NS5A region amplicationThe necleotide length of HVR1 and NS5A amplified by RT - PCR were 388bp and 573bp respectively, results of electrophoresis showed in Figl.2. HCV genotype classification14(70% ) of 20 patients were infected with HCV genotype II ( Ib), 4 (20% ) wereIII (2a) and 2 (10% ) were II mixed with III by the method of genotype specified PCR( Fig2). 12 patients infected with genotype II were also classified by direct sequencing. Only 1 of them was confirmed that he was infected with genotype 1c,the results of all the other patients(91.7% ) were no difference by the two methods.3. Relationship between HVR1 quasispecies heterogeneity and the effectiveness of IFN therapyThe quasispecies heterogeneity of HCV HVR1 of 3 responders and 4 nonre-sponders infected with genotype 1b was detected by the method of cloning andsequencing. 10 positive clonies of each sample were selected, the sequences of 63 clonies were abtained. The complexity of HVR1 quasispecies ( numbers of quasispecies) in the group of nonresponse was significantly higher than that in the group of response(t = 6. 737, p < 0. 01). The nucleotide diversity of HVR1 quasispecies was represented by the average mutant rate of each gene site. The results were similar to the complexity of HVR1 quasispecies (Table2).4. Th...
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