Department of Pathology, Division of Molecular Medicine and Medical Genetics, Kobe University Graduate School of Medicine, Kobe, Hyogo 6500017, Japan.
Centre for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Hyogo 6500017, Japan.
Mol Med Rep. 2018 Apr;17(4):5528-5537. doi: 10.3892/mmr.2018.8551. Epub 2018 Feb 2.
Hepatitis B virus (HBV) polymerase gene is targeted by nucleos(t)ide analogues (NUC), but it is unclear how HBV quasispecies of whole genome changes during early period of NUC treatment. To understand the unknown region of drug sensitivity and treatment resistance, HBV quasispecies of whole genome during early period of NUC treatment was examined using ultra‑deep sequencing. Eleven patients with chronic HBV infection who received NUC treatment were enrolled in the current study. Viral DNA was extracted from serum samples before and early period of NUC treatment. Polymerase chain reaction analysis was subsequently performed on the DNA products. The viral quasispecies of the entire genome was analyzed by ultra‑deep sequencing. The regions and positions corresponding to the changes in the quasispecies were investigated before and early period of NUC treatment. The secondary structure changes were predicted by mutations/substitutions detected using Lasergene Protean v14.1 software. The frequency of quasispecies variants increased significantly in the polymerase domain from before to early period of NUC treatment (3.08±1.28 vs. 3.51±1.47%, P<0.008), particularly the reverse transcription (RT) domain (3.76±1.25 vs. 4.52±1.37%, P<0.012). In addition, increased variation detected from HBsAg domain showed statistically significant during NUC treatment (6.81±3.26 vs. 7.81±3.26%, P<0.040). The amino acid (aa) mutations/substitutions were detected and compared from before to early period of treatment. Interestingly, most of them were located in the RT region (RT1 motif: aa21‑aa51) and small S region in the early duration of NUC treatment. Furthermore, several mutation patterns, such as cI97L and cP130T showed alterations in the secondary structure and predicted antigenicity of HBV protein. Although the HBV whole genome can be affected by NUC treatment, RT 1 motif region and small S region are more sensitive to the early period of NUC treatment. This study suggested the initial changes of HBV quasispecies might affect the long‑term drug sensitivity and resistance to NUC treatment.
乙型肝炎病毒 (HBV) 聚合酶基因是核苷 (酸) 类似物 (NUC) 的作用靶点，但在 NUC 治疗早期 HBV 全基因组准种如何变化尚不清楚。为了了解药物敏感性和治疗耐药性的未知区域，使用超深度测序研究了 NUC 治疗早期 HBV 全基因组准种。本研究纳入了 11 例慢性 HBV 感染接受 NUC 治疗的患者。从血清样本中提取病毒 DNA，然后对 DNA 产物进行聚合酶链反应分析。通过超深度测序分析全基因组病毒准种。在 NUC 治疗前和早期研究准种变化对应的区域和位置。使用 Lasergene Protean v14.1 软件检测突变/替换预测二级结构变化。从 NUC 治疗前到早期，聚合酶结构域中的准种变异频率显著增加 (3.08±1.28% vs. 3.51±1.47%，P<0.008)，尤其是逆转录 (RT) 结构域 (3.76±1.25% vs. 4.52±1.37%，P<0.012)。此外，HBsAg 结构域检测到的变异增加在 NUC 治疗期间具有统计学意义 (6.81±3.26% vs. 7.81±3.26%，P<0.040)。从治疗前和治疗早期检测到并比较了氨基酸 (aa) 突变/替换。有趣的是，它们中的大多数位于 RT 区 (RT1 基序：aa21-aa51) 和 NUC 治疗早期的小 S 区。此外，几种突变模式，如 cI97L 和 cP130T 显示 HBV 蛋白的二级结构和预测抗原性发生改变。尽管 HBV 全基因组可能受到 NUC 治疗的影响，但 RT1 基序区和小 S 区对 NUC 治疗的早期更为敏感。本研究表明，HBV 准种的初始变化可能影响 NUC 治疗的长期药物敏感性和耐药性。
