两例TTV感染者不同TTV克隆的DNA序列分析

DNA sequence analysis of different TTV clones of two cases with transfusion transmitted virus infection

目的探讨TTV的基因变异及其与致病性的关系。方法在ORF1区的高变区设计特异性引物建立巢式多聚酶链反应(PCR),扩增献血员与慢性非甲-庚型重型肝炎患者血清中的TTVDNA。PCR产物纯化回收后进行分子克隆,每例挑选10个不同TTVDNA克隆进行测序。不同TTVDNA克隆之间及其与日本株TA278之间进行序列比较并进行进化树分析。结果成功地构建了TTVDNA克隆。与G1a亚型TA278的序列相比较有74%～95%的同源。献血员存在2种不同的TTV病毒株,其序列有7%的差异,都为G1a亚型。慢性非甲-庚型重型肝炎患者中存在7种不同的TTV病毒株,彼此之间序列有5%～24%不同,分别属于G1a和G1b亚型。结论慢性非甲-庚型重型肝炎患者中TTV的基因变异比献血员中的复杂得多。TTV基因变异的复杂性及其基因变异过程中可能产生的强毒力病毒株均有可能在其致病机制中起一定作用。G1b亚型可能有一定的致病性。

[Objective] To investigate gene variant and the relationship between gene variant and pathogenicity of transfusion transmitted virus （TTV）. [Methods] A nested polymerase chain reaction （nested-PCR） assay with specific primers from high variable region （HVR） of open reading frame one （ORF1） of TTV genome was established, which was to amplify Trv DNA in the serum sample from a blood donor （BD） and a chronic non-A-G severe hepatitis patient （CSH） with TTV infection. The purified PCR product was cloned and 10 clones from each case were sequenced. The cloned TTV DNA sequences were compared with each other and with TA278 grouped Gla subtype. The sequences were also analyzed by Phylogentic tree. [Results] TTV DNA clone was successfully Constructed. The TTV DNA sequences shared 74%-95%homology with TA278. There were two different Trv strains in the blood donor and seven different Trv strains in the chronic non-A-G severe hepatitis patient. The Trv DNA sequences diverged from 5% to 24% in the CSH, but they were only 7% divergent in the BD. The Trv clones in the BD were all grouped Gla subtype, but they were grouped into Gla and Glb subtype in the CSH. [Conclusion] Gene variant of TTV was much more complicated in the chronic non-A-G severe hepatitis patient than that in the blood donor. The complexity of gene variant and the strong virulence strain delivered during gene variation are likely to be responsible for the pathogenesis of TTV. Trv strains grouped Glb maybe have pathogenicity to some extent.