Human liver tissues from 13 patients (6 male, 7 female; age, 31–46) with liver hemangioma receiving liver surgery were obtained from September 2011 to May 2014 in the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Patients were included in the present study if they exhibited normal biochemical indexes and had no history of hypertension, diabetes, fatty liver disease and other chronic diseases, including liver cirrhosis. Patients were excluded if they received hepatotoxic drugs, smoked and consumed alcohol in the first 3 months prior to surgery. The use of human tissue was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) and all patients gave informed consent prior to enrollment in the present study.

The D. salina strain. UTEX-1644, was obtained from the Algae Culture Collection at the University of Texas (Austin, USA) and were grown in modified PKS medium (NaCl 87.7 g/l; MgS04 with 7H₂0, 1.2 g/l; CaCl₂, 0.022 g/l; KN0₃ 1.0 g/l; KH₂P0₄, 0.054 g/l, ferric salt solution 2.0 ml/l including Na₂; EDTA with 2H₂ 0 0.74 g/l; FeCl₃, 6H₂0 0.216 g/l) with a 12-h light-dark cycle under a light intensity of 50 mmol photon m⁻²s⁻¹ at 26°C for 24 h (21). To amplify the IFN-α, IFN-γ and TA1 gene, the total RNA of human liver tissues was isolated using Trizol (Sigma, St. Louis, MO, USA). The IFN-α/IFN-γ fusion gene was generated using splicing by overlap-polymerase chain reaction (PCR) (22). The IFN-α/IFN-γ fusion gene and TA1 gene were then inserted into pUΩ-GUS using restriction enzyme HaeIII (New England BioLabs, Inc., Ipswich, MA, USA) and T4 DNA ligase (New England BioLabs, Inc., Ipswich, MA, USA) to generate pUΩ-IFN-α/IFN-γ and pUΩ-TA1 novel vectors, respectively. Subsequently, these vectors were co-transformed into D. salina cells using the glass bead method (21). The individual positive TDS colonies were selected using 3 mg/l of phosphinothricin (Hoechst-Roussel AG, Frankfurt, Germany) at 26°C for 5 days and used for further study after 24 h.

HepG2.2.15 cells purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were incubated in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and streptomycin, 380 mg/l antibiotic G-418 sulfate (Promega Corportation, Madison, WI, USA) and 1% L-glutamine at 37°C in 5% CO₂.

Cells were incubated in a 96-well plate at a density of 5×10⁴ cells per 100 µl at 37°C for 24 h. The cells were then treated with 1,000 IU/ml IFN-α or various concentrations of TDS (0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/ml) at 37°C for 5 days. Untreated cells were utilized as a control. Following treatment, cell viability was measured using an MTT assay as described previously (23). Based on the cell cytotoxicity detected by the MTT assay, the concentrations of TDS (0.4, 0.8 and 1.6 mg/ml) were selected for the following experiments in considerations of the lower toxicity. Furthermore, a treatment index (TI) was used to assess the clinical application prospect of the drug (24): TI <1, toxic, ineffective; TI=1-2, effective, with some toxicity; TI >2, greater effectiveness, with low toxicity.

Viral proteins in the culture medium, HBsAg and HBeAg, from the cells treated with different concentrations of TDS (0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/ml) were measured by using HBeAg (cat. no. KA3288) and HBsAg (cat. no. KA0286) ELISA kits (Abnova, Taipei City, Taiwan) according to the manufacturers' protocols.

HBV DNA was detected in HepG2.2.15 cells treated with IFN-α or TDS (0.8, 1.6 and 3.2 mg/ml) using quantitative PCR. Total DNA was extracted from the cell supernatant using the TIANamp Virus DNA/RNA kit (Tiangen Biotech Co., Ltd., Beijing, China) and Wizard^® Genomic DNA Purification kit (Promega Corporation). The quantification of HBV DNA copies was performed using the SYBR green premix reagent (Takara Biotechnology Co., Ltd., Dalian, China). Total DNA (2 µg) was used as the template for each quantitative PCR assay. PCR was performed using an ABI 7900 real-time PCR detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 93°C for 2 min, 10 cycles at 93°C for 45 sec and 55°C for 60 sec, and 30 cycles at 93°C for 30 sec and 55°C for 45 sec. GAPDH served as a control gene. The primers utilized for HBV DNA fragment amplification were as follows: Forward primer, 5′-CCTCTTCATCCTGCTGCT-3′ and reverse primer, 5′-AACTGAAAGCCAAACAGTG-3′. GAPDH primers: Forward primer, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse primer, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. Assays were repeated in triplicate and mean quantification cycle values were used to calculate the levels of HBV-DNA using the 2^−ΔΔCq method (25). The inhibitory rate was calculated using the formula: Inhibitory rate (%)=[DNA copy (C) control-C sample]/C control ×100.

A total of 3 day-old ducklings (weight, 40–50 g) were purchased from Zhejiang Academy of Agricultural Sciences (Zhejiang, China). The sex of ducklings was not distinguished as prior studies have demonstrated that this does not affect results (26,27). All ducks received ad libitum access to standard diet and water, and housed under controlled conditions (temperature, 28–30°C; humidity 56–70%; and a 24-h light cycle) (26,27). After adaptive maintenance for 3 days, 0.5 ml of blood was obtained from each duck for analysis. Only DHBV-positive ducklings were used for the subsequent experiments. Animal handling protocols were approved by the Animal Ethics Committees of The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China).

Ducks infected with congenital DHBV were randomly divided into 5 groups (each n=6): An IFN-α group treated with IFN-α (4,000 IU/injections, 500 µl/day; Sigma); 3 groups which received TDS via gastric perfusion at different concentrations (5, 10 and 20 g/kg TDS, respectively); and a control group treated with normal saline. The respective treatments were administered once daily for 21 consecutive days. Blood was drawn from all ducks prior to treatment (T0), following 7 (T7), 14 (T14) and 21 (T21) days of treatment and following withdrawal of the drug after 5 days (P5). The serum samples were separated using centrifugation at 7,000 × g for 15 min at 4°C and stored at −80°C. The levels of DHBV DNA in the serum were detected by quantitative PCR using the SYBR Green real-time PCR Master Mix (Takara Biotechnology Co., Ltd.) with the following primers: Forward primer, 5′-GATACTGGAGCCCAAACC-3′ and reverse primer 5′-GGCAGAGGAGGAAGTCAT-3′. GAPDH served as a control gene, forward primer, 5′-CACAGCCACACACGAAGACA-3′ and reverse primer, 5′-CCTTAGCCAGCCCCAGTAGA-3′. The thermocycling conditions were as follows: 95°C for 1 min, 40 cycles including 95°C for 5 sec, 56°C for 5 sec and 72°C for 25 sec and 40°C for 10 sec. The level of HBV-DNA was calculated using the 2^−ΔΔCq method (25).

Following 21 days of drug treatment, ducks were immediately anesthetized with sodium pentobarbital (Sigma; intraperitoneal injection; 150 mg/kg) and subsequently sacrificed by exsanguination. Liver specimens were collected from the ducks and separated into two sections (1×1 cm). One was fixed with 4% buffered formalin and embedded in paraffin for 24 h at room temperature. The samples were then stained with hematoxylin and eosin (H&E) for 15 min at room temperature and examined under an optical microscope.

The second section was also fixed with 4% buffered formalin for 24 h at room temperature and embedded in paraffin for 24 h at room temperature. Then the samples were stained with orcein for 30 min at 37°C and examined under an optical microscope to detect HBsAg.

Data are presented as the mean ± standard deviation and each experiment was performed in triplicate. Data were analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Statistically significant differences between two groups were detected using Student's t-test and the comparison of multiple groups was performed using one-way analysis of variance followed by a post-hoc Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

The effect of the drug treatment on cell viability and HBV antigen secretion in HepG2.215 cells in vitro was assessed. As presented in Fig. 1A, TDS had a marked inhibitory effect on cell viability, suggesting that TDS produced a cytotoxic effect on HepG2.2.15 cells. In addition, treatment with TDS (0.4, 0.8, 1.6 and 3.2 mg/ml) resulted in a significant reduction of HBsAg (Fig. 1B) and HBeAg secretion (Fig. 1C). The TI values of TDS for HBsAg and HBeAg were 2.96 and 3.07 respectively in HepG2.2.15 cells, indicating that TDS was effective and exhibited low toxicity (Fig. 1D). Furthermore, the TI values of TDS for HBsAg and HBeAg were significantly higher than that of IFN-α, which suggests that TDS may be more effective in clinical application.

To further confirm the anti-HBV activity of TDS, the levels of HBV-DNA in the cell supernatant were assessed. The results indicated that, compared with control group, treatment of TDS (0.8 and 1.6 mg/ml) significantly decreased HBV-DNA levels in the culture medium, with inhibition ratios of 49.3 and 65.7%, respectively (Fig. 2A and B). However, no significant difference was observed in the IFN-α group when compared with the control group. The results revealed that TDS treatment suppresses HBV-DNA replication in HepG2.2.15 cells.

Subsequently, the present study assessed the anti-HBV effect of TDS in vivo. As presented in Fig. 3A, duck serum DHBV-DNA levels were significantly decreased in the TDS group (20 g/kg) following treatment for 7, 14 and 21 days compared with that of the control group. Following treatment for 21 days and withdrawal of the drug for 5 days, the levels of duck serum DHBV-DNA in the TDS groups (10 and 20 g/kg) were significantly reduced when compared with the control group. Following drug withdrawal for 5 days, the levels of DHBV-DNA did not relapse in the TDS groups (10 and 20 g/kg). However, relapse following cessation of TDS was observed in the IFN-α and TDS (5 g/kg) groups.

Histological observation was then performed using H&E and orcein staining. The degree of swelling in liver cells was notable and the expression of infiltrating lymphocytes was positive in the control group. However, these results were alleviated following IFN-α and TDS treatment. In addition, HBsAg was observed as brown granules in the control group. However, a decrease in the signals for HBsAg was observed in IFN-α and TDS groups compared with the control group (Fig. 3B). These results demonstrated that TDS and IFN-α treatment alleviated inflammation and HBsAg in duck livers.