急性丙型肝炎患者病毒株部分基因克隆及其序列分析

Cloning and sequencing of partial genes of hepatitis C virus genome in patients with acute hepatitis C

目的探讨ＨＣＶ感染在肝炎发生中的病原学作用。方法对８９例急性肝炎进行ＨＣＶ标志物分析，并从ＨＣＶ ＲＮＡ阳性的５例非甲非乙型急性肝炎患者血清中提取ＨＣＶ ＲＮＡ，经随机引物逆转录合成ｃＤＮＡ，先以型别特异的ＰＣＲ 法分型，再用巢式ＰＣＲ扩增部分核心基因区序列，将产物连接Ｐ－ＧＥＭ－Ｔ载体，在大肠杆菌中表达后测序分析。结果非甲非乙型急性肝炎中ＨＡＶ、ＨＢＶ和ＨＣＶ阳性分别占４７．２％、２８．１％和１５．７％，ＨＡＶ和ＨＢＶ双重感染占 １４．６％，非甲、非乙和非丙肝炎占９％； ＨＣＶ分型显示Ⅱ型、 Ⅲ型和Ⅱ／Ⅲ混合型分别占８５８％、７１％和７１％； Ⅱ型血清用于序列分析，扩增序列４２４ｈＰ与原设计完全一致。急性肝炎株间核苷酸同源性在９８１％～９９５％。氨基酸同源性在９７．６％～９９．２％；前者的同源性在１型为９１．９０／０，在Ⅱ型为９４３％～９５．６％；后者与Ⅰ型、Ⅱ型的同源性在９２．３％～９５８％。结论ＨＣＶ为引起非甲非乙型急性肝炎的主要病毒之一，以Ⅱ型为主，应引起临床重视。

Objective To explore the etiological role of HCV in patients with acute hepatitis. Methods The prevalence of HCV infection in 89 patients with acute hepatitis was investigated by analysis of HCV RNA and HCV second generation antibody. HCV RNAs extracted from the sera of 5 patients with NANB acute hepatitis, which were positive for HCV RNA, were converted to cDNA by reverse transcription with random primer and genotyping by PCR with type-specific primers. The partial genes of HCV genome were amplified. The PCR products were expressed in E. colt with p-GEM-T vector, and their nucleotide sequences were determined by dideoxynucleotide chain-termination method. Results The inci- dence of hepatitis virus infection was 47.2% in HAV, 28. 1% in HBV and 15.7% in HCV, respectively. The incidence of HAV and HBV coinfection was 14.6% and the rate of non-A, non-B and non-C hepatitis was 9% in all patients. The geno- type of Hey-RNA positive patients was 85.8% in Hey-Ⅱ, 7. 1% in HCV-Ⅲ and 7.1% in combining Hey- Ⅱ /Ⅲ, respectively. The partial sequence of HCV genome in 5 patients with non-A and non-B acute hepatitis was amplified and the fragment was 424 hp in accordance with original design. The homology of the sequences was 98.1%-99.5% in nucleotide acid and 97.6%-99.2% in amino acid among five isolates. The average homology was 91.9% or 94.3%-95.6% for nucleotide sequences between Hey-Ⅰ or Hey-Ⅱ and the 5 isolates, and 92.3%-95.8% for amino acid sequences between the 5 iso- lates and HCV- Ⅰor HCV- Ⅱ, respectively. Conclusion HCV infection is one of the main hepatitis viruses in patients with acute hepatitis, in which the Hey-Ⅱ genotype is dominant and should be paid attention to it.