拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测

Variations in hepatitis B virus polymerase responsible for lamivudine resistance

目的建立拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测方法，对该部位变异类型、发生率及其与病毒滴度的关系进行初步分析。方法拉米夫定治疗的慢性乙型肝炎患者164例，在其中治疗64周时HBVDNA阳性的101份血清中，选择9份血清进行分析。设计台成一对引物SI、A1，采用聚台酶链反应法扩增得到包括YMDD基序（motif）的部分P区基因，片断长926bp，对扩增片断的部分校苷酸（约500bp）进行序列分析。HBVDNA定量用分枝DNA信号扩增祛。结果在9例患者中，发现YMDD（酪氨酸－蛋氨酸－天门冬氨酸－天门冬氨酸）基序变异的5例，占56％，其中M（蛋氨酸）－I（异亮氨酸）3例，M（蛋氨酸）－V（缬氨酸）2例，后者合并D（天门冬氨酸）→G（甘氨酸）变异1例。在2例血清HBVDNA低滴度（＜100MEq／ml）患者中未发现YMDD基序变异，7例中、高滴度（＞300ME／ml）患者中5例发现YMDD基序变异，占71％。结论拉米夫定是一种新的治疗慢性乙肝较有潜力的抗病毒药物，若能在治疗过程中对HBVDNA滴度变化及病毒变异发生进行监测，可指导临床的合理用药及联台用药。

Obectives To investigate variations in hepatitis B virus (HBV) polymerase gene in chronic HBV infected patients resistant to lamivudine therapy. Methods Partial segments of HBV DNA polymerase gene were amplified by polymerase chain reaction (PCR) from nine patients with chronic HBV infection and who were resistant to lamivudine therapy. The analysis of nucleotide sequence was performed on an Applied 373 automated sequencer. Meanwhile, titre of HBV DNA was measured by branched-DNA assay (Chiron). Results of nine patients with HBV DNA positive after 64 weeks of treatment, five (56%) had variations in the highly conserved YMDD motif in domain C of the HBV polymerase, three of those were substitution of isoleucine(I) for methionine(M), two were substitution of valine (V) for methionine(M). Additionally, in two patients with variations characterized by substitutions of V for M, one had a simultaneous amino acid chance from the first aspartic acid to glycine and this pottern of variation was not roprted in other literatures. With resect to viremia, in two subjects with lower titre of HBV DNA (<100MEq/ml), no variation was found in YMDD motif, whereas in seven Patients with higher titre of HBV DNA (>300MEq/ml), five(71%) had variations in the YMDD motif. Conclusions Lamivudine is a potent anti-viral agent for the treatment of chronic HBV infection. It is likely that resistance to lamivudine is caused by the variations in the YMDD motif of the HBV polymerase gene.[