慢性乙型肝炎患者抗病毒治疗中的病毒准种演变

目的 探讨拉米夫定耐药后换用恩替卡韦补救治疗的慢性乙型肝炎患者的病毒准种演变.方法 提取1例慢性乙型肝炎患者治疗中的7个不同时间点(0、24、48、60、72、96、152周)血清中的HBV DNA,巢式聚合酶链反应法扩增HBV DNA聚合酶基因逆转录酶区,克隆测序法对逆转录酶区氨基酸替换形式及准种分布进行分析,并采用扩增耐药突变系统聚合酶链反应法对患者病毒种群中野毒株与病毒总量进行定量检测.结果 患者在治疗过程中主要存在rtM204V、rtL180M+rtM204V和rtM204I 3种拉米夫定耐药相关的病毒株变异形式,各病毒株所占比例不断发生变化,基线时HBV野毒株为优势病毒株,在病毒学突破时,种群中全部为耐药突变株;换用恩替卡韦治疗后,随着病毒载量的下降,拉米夫定耐药突变株被抑制,野毒株在种群中比例逐渐上升,并成为优势病毒株(79.3%).扩增耐药突变系统聚合酶链反应检测结果显示,在基线和发生病毒学突破时,野毒株在种群中的比例分别为68.55%和0.21%,换药治疗后24周,野毒株比例开始上升,此后野毒株占种群的比例波动于16.01%～26.93%.结论 慢性乙型肝炎患者在核苷(酸)类药物序贯治疗过程中,HBV种群的准种分布一直处于动态变化中.不同的HBV准种演变模式可能在恩替卡韦补救治疗中对恩替卡韦耐药发生的作用也不相同.

Abstract：

Objective To investigate the evolution of hepatitis B virus (HB V) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment. Methods Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96,152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR). Results Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%. Conclusions The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.

Evolution of hepatitis B virus quasispecies during antiviral therapy in one chronic hepatitis B patient

Objective To investigate the evolution of hepatitis B virus (HB V) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment. Methods Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96,152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR). Results Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%. Conclusions The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.