Laboratory assessments

Routine physical examinations with biochemical and hematologic assessments were conducted at weeks 0, 12, 24, 48, and 72. HBV RNA was extracted from patient serum samples (200 μL) using a nucleic acid extraction kit (Sansure Biotech Inc. China) which was developed based on the magnetic bead technology [17]. Eluted HBV RNA (10 μL) obtained from HBV RNA extraction was used for reverse transcription. Primers of HBV RNA-RACE, HBV RNA-forward, HBV RNA-reverse, and HBV RNA-probe (see Supplementary) that target conserved regions of the HBV genome were obtained from the literature [12]. Armored RNA internal controls were added during sample lysis [18]. In the absence of DNA polymerase and cDNA primers, HBV RNA was reverse transcribed into cDNA under the temperature of 50 °C for 30 min. After adding DNA polymerases and cDNA primers, the cDNA amplification was performed by an activation step at 95 °C for 2 min, followed by 50 two-step cycles (each cycle 15 s at 95 °C and 30 s at 60 °C), and a cooling step down to 25 °C for 10 s. The fluorescence of cDNA was detected and measured by the 7500 Real-Time PCR System (Applied Biosystems^®). More details are provided in our Supplementary.

Elecsys tests (Roche Diagnostics GmbH, Germany) were used to measure HBeAg, anti-HBe, HBsAg, and anti-HBs. HBV DNA was quantified by the Roche Diagnostics Cobas^® Amplicor HBV Test, Version 2.0 (Roche Diagnostics, Germany). ALT assay was conducted using Architect c8000 clinical chemistry analyzer (Abbott, USA) with the IFCC standard for enzyme determination. The NCBI HBV genotyping tool [19] was applied to determine HBV genotypes with inputs of HBV polymerase sequences extracted by conventional Sanger sequencing. Detection limits of HBV RNA, HBV DNA, HBeAg, and HBsAg were 250 copies/mL, 20 IU/mL, 1 COI (cut of index), and 0.05 IU/mL, respectively. The log₁₀ values of four biomarkers above were transformed prior to statistical analyses.