HBV准种变异与阿德福韦酯治疗HBeAg阳性慢性乙型肝炎持续疗效的关系

目的观察阿德福韦脂治疗HBeAg阳性慢性乙型肝炎患者HBV聚合酶（HBV-P）基因序列的准种组成及变异特点,探讨其与抗病毒疗效的关系。方法巢式PCR法扩增HBV-P基因中覆盖B区-E区序列的基因片段,PCR产物纯化后直接测序。用DNASTAR软件的CLUSTALV方法对HBVDNAP基因片段的核苷酸和氨基酸差异、变异类型进行分析,以评估HBV准种复杂性。128例HBeAg阳性慢性乙肝患者予阿德福韦酯10mg,每日一片口服,抗病毒治疗48-96周,疗效评估包括HBVDNA血清学、HBeAg/HBeAb血清学转换及生化学应答。观察阿德福韦酯治疗完全应答组、部分应答组和无应答组病人治疗24周、48周、96周的准种组成特点,并分析其与疗效的关系。结果 128例患者应用阿德福韦酯治疗两年,完全应答23例,部分应答86例,无应答19例。HBVDNA转阴率50.8%（65/128）,HBeAg血清转阴率为21.1%（27/128）,ALT复常率为60.9%（78/128）。7例患者产生了病毒变耐药变异,其中4例为rtn236T变异,2例为rtA181V变异,1例为rtN236T和rtA181V联合变异,耐药率为5.4%。所有患者HBV准种变异在治疗24周、48周、96周时均具有不同程度的改变,无应答组24周与48周时HBV准种复杂性显著高于完全应答组（P〈0.001）。结论慢性乙型肝炎患者血清HBVP区存在准种现象,且该区准种随宿主病程进展可发生动态变化。HBVP区准种变异的复杂性及异质性与慢性乙型肝炎患者阿德福韦酯抗病毒治疗的持续疗效存在负向相关性。

Relationg of HBV P gene quasispecies mutants and adefovir dipivoxil in the treatment of HBeAg-positive chronic hepatitis B

Objective To investigate the relationship between hepatitis B virus P gene （HBV-P） quasispecies mutants and adefovir dipivoxil in the treatment of HBeAg-positive chronic hepatitis B.Methods Nested-PCR（NT-PCR）amplification of HBV-P gene in the coverage zone B zone-E gene fragment sequences,PCR product was purified and directly sequenced to assess the HBV quasispecies complexity.The difference of amino acid of part P gene in HBV DNA was analyzed,and the mutation was classified by using CLUSTAL V method with DNA STAR software.128 patients received adefovir dipivoxil 10 mg once daily for 24 to 96 weeks.Therapeutic effect evaluation involved the serum HBV DNA level,HBeAg sero-conversion and biochemical response.Analysis the relationship between HBV-P quasispecies and nucleoside analogues treatment effect among patients with the long term complete responders （CRL）,part responders （PR） and non responders （NR） at the onset,48 weeks and 96weeks of adefovir dipivoxil treatment.Results Of 128 patients received adefovir dipivoxil for two years,23 were CRL,86 were PR,and 19 were NR.Normal alanine amino transferas （ALT） was seen in 61.4 % patients（78/128）;HBeAg sero-conversion in 21.9% patients（27/128）;and HBV DNA negative in 50.8%（65/128）.7 patients had resistance mutation,4 were rtn236T,2 were rtA181V,and another were rtN236T and rtA181V resistance mutation.The complexity and heterogeneity of quasispecies of this region were higher in adefovir dipivoxil treatment than no treatment,and so in the non responders groups than in long term complete responders.All the quasispecies were different among the three groups during treatment of adefovir dipivoxil.The quasispecies mutation was significantly different between the group CRL and NR on the 24th,48th week of treatment （P0.001）.Conclusion There is a quasispecies nature of HBV-P gene in CHB patient.The quasispecies of this region may change dynamically with the changes of activity of hepatitis in the CHB patient.The complexity and heterogeneity of quasispecies of this region were low at the begin time points of adefovir dipivoxil treatment,while they became relatively high after a certain period of treatment and gradually went higher as time accumulates.The complexity of HBV quasispecies was correlated to adefovir dipivoxil in the treatment HBeAg-positive chronic hepatitis B.