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Early changes in quasispecies variant after antiviral therapy for chronic hepatitis B.

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.

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