150例慢性丙型肝炎患者HCV基因型和NS3蛋白酶编码区序列的分析

Analyses of HCV genotypes and NS3 protease-encoding region of 150 patients with chronic hepatitis C

目的对来源于我国不同地区的150例慢性丙型肝炎患者进行HCV基因分型和NS3蛋白激酶编码区序列分析。方法基因分型采用基因芯片法;NS3区序列扩增采用自行设计的型特异性引物介导巢式RT-PCR,序列分析采用PCR产物直接测序。结果 150例患者HCV基因分型结果为1b占66.7%(100/150),2a占22.7%(34/150),3a占4%(6/150),3b占4%(6/150),6型占2%(3/150),1a占0.7%(1/150)。对NS3区序列采用进化树分析进行分型,其结果与基因芯片法分型结果高度一致,主要基因型(1a、2b)的NS3区序列无明显地区性聚集倾向。未检出与新型HCV蛋白酶抑制剂telaprevir耐药相关的V36M/A、T54A、R155K/T和A/156V/T变异。结论我国慢性丙型肝炎患者仍以1b型感染为主,采用NS3蛋白酶编码区序列分析可同时进行基因分型和针对蛋白酶抑制剂的耐药变异检测,我国天然存在的相对优势变异株发生telaprevir原发耐药的可能性很小。

Objective To investigate the HCV genotypes and the sequences of NS3 protease-encoding region of 150 patients with chronic hepatitis C originated from various districts of China. Methods Phylogenetic tree analysis based on NS3 sequence was performed for determination of HCV genotypes, and the results were compared with that obtained from gene chip. A nested RT-PCR assay with sell designed type-specific primers was developed and used for amplification of viral NS3 region from sera of 150 patients. Direct PCR sequencing, if necessary, was performed to analyze the sequences. Results The HCV genotyping of 150 patients showed that lb genotype was predominant and of 66. 7% fraction （100/150）. The prevalence of 2a, 3a, 3b, 6 and la genotypes were 22. 7% （34/150）, 4% （6/ 150）, 4% （6/150）, 2% （3/150） and 0. 7% （1/150）, respectively. The genotyping results obtained with NS3 sequence as phylogenetic tree were in high concordance with that obtained by gene chip. No obvious regional cluster inclination was observed in NS3 sequences of lb and 2a, the major genotypes. The variations V36M/A, T54A, R155K/T and A/156V/T, contributed to the resistance against telaprevir, a new HCV protease inhibitor, were not detectable. Conclusions HCV genotype lb is still predominant at present in Chinese patients with chronic hepatitis C. Sequencing of N.S3 protease-encoding region is a practical assay in both HCV genotyping and detecting the variations against HCV protease inhibitors. Few opportunity of primary telaprevir resistance caused by primary resistant variations of the virus exists in Chinese HCV patients.