摘要
Genotyping of hepatitis C virus (HCV) is important because of its clinical and epidemiological implications. The distribution of HCV genotypes in China with a simple and practical HCV typing method according to program ABC was reported. The method is based on restriction fragment length polymorphism (RFLP) of the amplified DNA from the 5′ non-coding region of the genome. Instead of digestion of each sample with all the restriction enzymes, the samples were first divided into five groups in program A (BsrBⅠ, HaeⅡ, HinfⅠ) and then were further typed in program B (BstUⅠ) and C (HaeⅢ). The new method was used for genotyping 187 isolates from patients of different regions of China. HCV genotypes 1a (1.1%), 1b (67.4%), 2a (12.8%), 2b (3.2%), 3b (5.4%), 6a (0.5%), 1b+2a (5.4%), 2a+2b (2.1%) and 1b+2b (2.1%) were identified and confirmed by phylogenetic analysis. Chosing 3 strains randomly from the 10 samples of genotype 3b detected. The 3 strains were subjected to A-T clone and sequencing. The sequencing results indicated that the homology among the 3 strains is 99.54%～100%. Comparison of the homology between strain KQ50 and the standard strains in Genbank showed that strain KQ50 had the highest homology with genotype 3b, which confirmed the reliability of our genotyping method further. Nucleotide sequences obtained for the first time in this study have been submitted to Genbank. The accession numbers are respectively Ay311400, Ay311401, Ay311402 (3b of 5′-NCR), Ay443346, Ay 443347 (3b of NS5B), Ay443348 (1a of 5′-NCR). 147 HBV genotype 1b serum samples were selected from HCV infected in- and out patients in our hospital. The amplified DNA fragment from 5′ non-coding region was digested with restriction endonuclease Mbo I and BamH I. As a result, 80(54.42%)samples have one MboⅠsite, 6 samples (4.08%) have two Mbo I sites, 17(11.56%) samples have one BamHⅠrestriction site, 26(17.69%) have neither BamH I nor Mbo I restriction site and 18 (12.24%) have both strains with MboⅠsite and those without MboⅠsite. 6 strrains were randomly selected from those with one BamHⅠrestriction site. The 6 strains were sequenced and compared with 39 standard strains of HCV genotype 1b. The sequencing reports confirmed that the strains of HCV genotype 1b created BamHⅠrestriction site by a substitution of nucleotide at position 117. Phylogenetic analysis showed that the 6 strains belong to the same division in genetic distance, however, differ from the standard strains of genotype 1b. Whether the 6 strains are the other subtypes of genotype 1 and the mutation of genotype 1b in 5′-NCR at position 117 exists only in China are not sure yet. It is also noticeable that this mutation might have relationship with response to alpha-interferon treatment and play an important role in chronic hepatitis C.;
Genotyping of hepatitis C virus (HCV) is important because of its clinical and epidemiological implications. The distribution of HCV genotypes in China with a simple and practical HCV typing method according to program ABC was reported. The method is based on restriction fragment length polymorphism (RFLP) of the amplified DNA from the 5′ non-coding region of the genome. Instead of digestion of each sample with all the restriction enzymes, the samples were first divided into five groups in program A (BsrBⅠ, HaeⅡ, HinfⅠ) and then were further typed in program B (BstUⅠ) and C (HaeⅢ). The new method was used for genotyping 187 isolates from patients of different regions of China. HCV genotypes 1a (1.1%), 1b (67.4%), 2a (12.8%), 2b (3.2%), 3b (5.4%), 6a (0.5%), 1b+2a (5.4%), 2a+2b (2.1%) and 1b+2b (2.1%) were identified and confirmed by phylogenetic analysis. Chosing 3 strains randomly from the 10 samples of genotype 3b detected. The 3 strains were subjected to A-T clone and sequencing. The sequencing results indicated that the homology among the 3 strains is 99.54%～100%. Comparison of the homology between strain KQ50 and the standard strains in Genbank showed that strain KQ50 had the highest homology with genotype 3b, which confirmed the reliability of our genotyping method further. Nucleotide sequences obtained for the first time in this study have been submitted to Genbank. The accession numbers are respectively Ay311400, Ay311401, Ay311402 (3b of 5′-NCR), Ay443346, Ay 443347 (3b of NS5B), Ay443348 (1a of 5′-NCR). 147 HBV genotype 1b serum samples were selected from HCV infected in- and out patients in our hospital. The amplified DNA fragment from 5′ non-coding region was digested with restriction endonuclease Mbo I and BamH I. As a result, 80(54.42%)samples have one MboⅠsite, 6 samples (4.08%) have two Mbo I sites, 17(11.56%) samples have one BamHⅠrestriction site, 26(17.69%) have neither BamH I nor Mbo I restriction site and 18 (12.24%) have both strains with MboⅠsite and those without MboⅠsite. 6 strrains were randomly selected from those with one BamHⅠrestriction site. The 6 strains were sequenced and compared with 39 standard strains of HCV genotype 1b. The sequencing reports confirmed that the strains of HCV genotype 1b created BamHⅠrestriction site by a substitution of nucleotide at position 117. Phylogenetic analysis showed that the 6 strains belong to the same division in genetic distance, however, differ from the standard strains of genotype 1b. Whether the 6 strains are the other subtypes of genotype 1 and the mutation of genotype 1b in 5′-NCR at position 117 exists only in China are not sure yet. It is also noticeable that this mutation might have relationship with response to alpha-interferon treatment and play an important role in chronic hepatitis C.;
出处
《中国生物工程杂志》
CAS
CSCD
2004年第9期93-94,共2页
China Biotechnology
