干扰素治疗早期乙型肝炎病毒的准种进化

Early evolution of hepatitis B virus quasispecies during IFN-α treatment in patients with chronic hepatitis B

目的应用生物信息学方法研究干扰素-α(IFN-α)治疗早期乙型肝炎病毒(HBV)全基因组准种进化及其与病毒学应答的关系。方法对16例进入免疫清除期的慢性乙型肝炎患者进行IFN-α抗病毒治疗,治疗12周有6例取得完全病毒学应答,3例部分病毒学应答,7例无应答。分别采集治疗前和治疗12周的患者血清,提取HBV DNA后扩增HBV全基因组,进行分子克隆后测序,对得到的序列进行生物信息学分析从而研究病毒的准种进化。结果除了乙肝病毒前C/C基因,在IFN-α治疗早期无其他基因准种特征的动态变化。治疗前完全应答组前C/C基因氨基酸水平的平均遗传距离[2.868(1.727~5.411)10^(-2)替换数/位点]大于无应答组[0.917(0.449~3.695)10^(-2)替换数/位点](P<0.001);部分应答组[1.904(1.272~2.349)10^(-2)替换数/位点]大于无应答组[0.917(0.449~3.695)10^(-2)替换数/位点](P=0.006)。治疗前完全应答组前C/C基因平均非同义替换率[1.410(0.804~3.188)10^(-2)替换数/位点]大于无应答组[0.293(0.205~0.651)10^(-2)替换数/位点](P=0.008);治疗12周部分应答组氨基酸水平平均遗传距离[1.755(0.974~2.172)10^(-2)替换数/位点]大于无应答组[0.869(0.276~1.475)10^(-2)替换数/位点](P=0.047);部分应答组与无应答组在IFN-α作用下病毒进化速率差异无统计学意义(P=0.53)。结论慢性乙型肝炎免疫清除期,前C/C基因氨基酸水平准种多样性高的患者在IFN-α治疗早期更容易取得病毒学应答。

Objective To study the dynamic change of hepatitis B virus（HBV）genomic quasispeices during the early stage of IFN-αtreatment and its impact on virological response. Methods Serum HBV DNA levels were extracted,amplified,cloned and sequence analysis were done for hepatitis B patients treated with INFalpha at baseline and weeks 12. Results A total of 16 patients with chronic hepatitis B were recruited.Six patients acquired complete virological response,3partial and 7no responses.There were no significant heterogeneity in genomic quasispecies（except pre-C ORF）among these 3groups of patients with different status of virological responses after 12 weeks of treatment.For pre-C gene,the mean genetic distance at amino acid levels of responders [2.868（1.727-5.411）10^（-2） substitution/site]was greater than that of non-responders[0.917（0.449-3.695）10^（-2） substitution/site]（P〈0.001）,partial responders [1.904（1.272-2.349）10^（-2）substitution/site]was greater than that of non-responders [0.917（0.449-3.695）10^（-2） substitution/site]（P=0.006）;the dN of responders [1.410（0.804-3.188）10^（-2） substitution/site]was greater than that of non-responders [0.293（0.205-0.651）10^（-2） substitution/site]（P=0.008）.The mean genetic distance at amino acid levelsof partial responders [1.755（0.974-2.172）10^（-2） substitution/site]was significantly higher than that of the non-responders at week 12 [0.869（0.276-1.475）10^（-2） substitution/site]（P=0.047）.There was no significant difference in the viral evolutionary rates between partial responders [0.7（0.1-13.94）×10^-4 substitution/site·month]and non-responders [1.41（0.04-13.3）×10^（-4） substitution/site·month]（P=0.53）. Conclusions At the immunoclearance phase of chronic hepatitis B,patients with greater viral quasispecies diversity in the HBV pre-C gene tend to obtain faster early virological responses for IFN-αtreatment.