The patient was a 49-year-old Chinese female with hepatitis B e antigen-positive chronic HBV infection. Initially, she received lamivudine monotherapy (100mg per day, p.o.) and had serum levels of HBV DNA of 8.23log10copies/mL. After receiving lamivudine for five months, the viral load decreased to 4.39log10copies/mL, and then became undetectable after an additional three months. Unfortunately, HBV DNA rebounded to 6.86log10copies/mL and rose continuously in the next two months. At this point, signature mutations of lamivudine resistance (rtL180M/M204V) were detected by DNA sequencing of the reverse transcriptase domain of circulating HBV, and the genotype was of clone B (Invitrogen, Shanghai, China). Blood samples were collected after viral breakthrough.

Lamivudine was obtained from GlaxoSmithKline (Suzhou, China). Adefovir (GlaxoSmithKline, Tianjin, China) was used as a positive control for inhibition of the lamivudine-resistant HBV strain. NCC (Fig. 1) with 98.5% purity (as determined by high-performance liquid chromatography) was designed and synthesized in our laboratory (The College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou). Stock solutions (10mM) of the three compounds were dissolved in phosphate-buffered saline.

Fig. 1.

Chemical structure of NCC.

Molecular formula: C12H15FN6O4. Molecular weight: 326.28g/mol.

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HBV DNA was extracted using a Viral DNA Extraction kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions. Briefly, patient serum was digested by OB protease and Buffer BL with linear acrylamide and RNase A, and then incubated sample at 65°C for 10min, followed by lysate with absolute ethanol (room temperature, 96–100%). The lysate was transferred into a HIBind DNA mini column and cleared of liquid by centrifugation at 8000×g for 1min to bind DNA. The HIBind DNA mini column was washed by Buffer HB and DNA wash Buffer and cleared of liquid by centrifugation at 15,000×g for 2min to dry the column. After incubated the column by preheated Elution Buffer for 5min at room temperature, DNA was eluted by centrifugation at 8000×g for 1min. Polymerase chain reaction (PCR) amplification of the full-length HBV genome was performed according to the methods of Günther et al.,17 with the primers p1 and p2 (shown in Table 1). Cycling parameters: pre denaturation 94°C for 5min; 94°C for 40s, 60°C for 78s, 68°C for 3min, the extension time plus 1min after every 10 cycles, 30 cycles total; final additional extension at 72°C for 10min; hold at 4°C. Amplicons were cloned into pMD18-T (TaKaRa Bio, Dalian, China) to generate the T-HBV recombinant for sequencing the reverse transcriptase domain. Results showed that the cloned HBV genome harbored rtL180M/M204V mutations, and that the genotype was clone B.

HBV DNA is circular and partially overlapped, and its genome comprises four open reading frames named S, C, P, and X. According to this feature of HBV genome structure, we designed and amplified four fragments designated A, B, C, and D, and constructed a recombinant plasmid harboring 1.3 units length of HBV genome (4080bp) to ensure efficient HBV replication and expression. Fragment A is located from nt 2846 to nt 3215 of HBV genome, fragment B is located from nt 1 to nt 1393 of HBV genome, fragment C is located from nt 1394 to nt 3215 of HBV genome, and fragment D is located from nt 1 to nt 495 of HBV genome (A+B+C+D=4080bp) (Table 1). T-HBV recombinant was used as a template for amplification of A, B, C, and D gene fragments using the corresponding primers (shown in Table 1) with the following reaction procedure: 94°C for 4min, then 30 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1min, and 72°C for 6min. The A+B and C+D gene fragments were obtained by recombinant PCR with the primers (shown in Table 1) and A, B and C, D gene fragments, respectively, as templates, and the amplification condition was the same as above. Fragment A+B was then digested with HindIII and EcoRI, and fragment C+D was digested with EcoRI and NotI. A 1.3-fold-over- length HBV genome containing fragments A+B and C+D was then ligated into HindIII and NotI cleaved pcDNA 3.1 plasmids by T4 DNA Ligase at 16°C for 16h, resulting in pcDNA3.1-1.3HBV.

The human HepG2 was obtained from the China Center for Typical Culture Collection (Wuhan, China). Cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco, New York, NY, USA). HepG2 cells were transfected with pcDNA3.1-1.3HBV encoding mutant HBV using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Two days after transfection, cells were selected in medium supplemented with 600μg/mL G418 (Sigma–Aldrich, Saint Louis, MO, USA) for three weeks and renewed every three days. The G418-resistant clone was transferred to a T25 culture flask and maintained in the same Dulbecco's modified Eagle's medium with G418 at 300μg/mL. The novel stable cell line was designated “HepG2.RL1 cell.” The surface antigen of HBV (HBsAg) and e antigen of HBV (HBeAg) in cell culture supernatants were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Kehua Bio-engineering, Shanghai, China), and intracellular HBV DNA was extracted from the cells by Tissue viral DNA Extraction Kit (Omega Bio-Tek, USA) according to the manufacturer's instructions, respectively. The amount of intracellular and extracellular HBV DNA were determined by quantitative real-time PCR (qPCR) based on the TaqMan technology and performed in a Light-Cycler (Roche, Mannheim, Germany) with the HBV Fluorescent Quantitative PCR Detection Kit (Qiagen CO., Ltd. Shenzhen, China) following the manufacturer's protocol. After an initial denaturation at 95°C for 90s, 40 cycles of denaturation at 94°C for 5s and annealing/extension at 56°C for 45s were conducted.

Cell medium (30mL) was collected and centrifuged at 3000×g for 30min at room temperature. The supernatant was filtered through a 0.22μm filter membrane. HBsAg (+) healthy blood serum (4mL) was diluted 1:5 with PBS and also filtered through a 0.22μm filter membrane. Subsequently, filtrates were mixed at 1:1 and incubated overnight at 4°C, then was centrifuged and resuspended in culture medium. Suspensions were placed on a copper network supported by carbon membranes. They were examined under immuno-transmission electron microscopy (ITEM) after negative staining with 2% phosphotungstic acid for observation of virus particles.

HepG2.2.15 cell line (wild-type HBV in the stable HBV-producing) was obtained from the Wuhan Institute of Virology (Wuhan, China) and maintained in the same manner as HepG2 cells except that 200μg/mL G418 was added to the culture medium in our laboratory. Compound cytotoxicity was determined by the MTT assay on HepG2.2.15 cells and HepG2.RL1 cells.18 Cells were treated with lamivudine, adefovir, and NCC at various concentrations (62.5, 125, 250, 500, and 1000μM).

HepG2.2.15 cells and HepG2.RL1 cells were seeded in 24-well culture plates at 3×104 cells per well and cultured for 24h. Further, lamivudine and adefovir at various concentrations and NCC (0.04, 0.2, 1, 5μM) were added to cells for six days. On day 6, HBV DNA was quantified as described previously by qPCR to assess the inhibitory efficiency of drugs on viral replication. HBsAg and HBeAg production were assessed by ELISA. The 50% effective concentration (EC50) was calculated by regression analyses. Fold resistance was calculated as the ratio of mutant EC50 to wild-type EC50.

Statistical analyses were performed using SPSS v17.0 (IMB, Armonk, NY, USA). Results are the mean±SD. One-way ANOVA was used to determine the statistical significance of differences between groups. p<0.05 was considered significant.

As shown in Fig. 2A, the full-length HBV genome was extracted from the patient serum and amplified by PCR. The amplicon was cloned into pMD18-T, and used as a template for amplification of A, B, C, and D gene fragments. The PCR products were purified with Gel Extraction Kit (Omega Bio-Tek, USA) from an agarose gel, with a single strip (Fig. 2B). The A+B and C+D gene fragments were obtained by recombinant PCR, and digested with HindIII and EcoRI, EcoRI and NotI, respectively, then purified with Gel Extraction Kit (Fig. 2C). The gel purification of double-enzyme A+B and C+D gene fragments were ligated into the Hind III and Not I-digested pcDNA 3.1 plasmid (Fig. 2D), producing a recombinant clone termed pcDNA3.1-1.3HBV (Fig. 3). For restriction double-enzyme digestion by Hind III and Not I, the recombinant plasmid pcDNA3.1-1.3HBV was separated into two bands with pcDNA 3.1 plasmid (5428bp) and A+B+C+D (4080bp) (Fig. 2E), as well as digestion by Hind III, the 9.5kb linear plasmid pcDNA3.1-1.3HBV was shown (Fig. 2F). The full sequence of cloned pcDNA3.1-1.3HBV was uploaded as a supplementary file (Doc.1).

Fig. 2.

The PCR products of agarose gel electrophoresis analysis.

(A) Full-length HBV genome amplified by PCR. Lane M: 250bp DNA Ladder Marker; Lane 1: the full length HBV genome (3215bp). (B) Gel purification of A, B, C and D PCR fragments. Lane M: DL2000 DNA Marker; Lane 1, 2, 3,4: gel purification of A (370bp), B (1393) bp, C (1822bp), and D (495bp) PCR fragment, respectively. (C) Gel purification of A+B and C+D restriction enzyme digestion fragments. Lane M: 500bp DNA Ladder Marker; Lane 1: gel purification of A+B fragment digested by Hind III and EcoR I; Lane 2: gel purification of C+D fragment digested by EcoR I and Not I. (D) Gel purification of double-enzyme digested pcDNA 3.1 plasmid. Lane M: DL15000 DNA Marker; Lane 1: gel purification of double-enzyme pcDNA 3.1 plasmid (5428bp) digested by Hind III and Not I. (E) Restriction double-enzyme digestion identification of recombinant plasmid pcDNA3.1-1.3HBV by Hind III and Not I. Lane M: DL15000 DNA Marker; Lane 1: digestion of recombinant plasmid pcDNA3.1-1.3HBV by Hind III and Not I as pcDNA 3.1 plasmid (5428bp) and A+B+C+D (4080bp). (F) Enzyme digestion identification of recombinant plasmid pcDNA3.1-1.3HBV by Hind III. Lane M: DL15000 DNA Marker; Lane 1: digestion of plasmid pcDNA3.1by Hind III; Lane 2: digestion of recombinant plasmid pcDNA3.1-A+B by Hind III; Lane 3: digestion of recombinant plasmid pCDNA3.1-1.3HBV by Hind III.

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Fig. 3.

Construction of the pcDNA3.1-1.3HBV recombinant plasmid.

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To further characterize HepG2.RL1 cells, the supernatants and cells of each generation were collected for 100 continuous passages. HBeAg and HBsAg were secreted continuously at a stable level (Fig. 4A). The level of HBeAg produced by HepG2.RL1 cells was higher than the level of HBsAg under identical conditions. Amounts of viral DNA increased markedly at the 20th generation during the culture period. At the 10th and 100th generations, HepG2.RL1 cells produced 5.67 and 8.08log10copies/mL of intracellular DNA, as well as 4.51 and 5.61log10copies/mL of extracellular DNA, respectively (Fig. 4B).

Fig. 4.

Time-course of secretion of viral antigens and HBV DNA, and electron micrograph of HBV particles in the HepG2.RL1 cell line

(A) Levels of HBeAg and HBsAg in the culture supernatants of the HepG2.RL1 cell line according to generation. OD450/630, optical density at 450nm and 630nm. (B) Quantity of intracellular and extracellular DNA in HBV according to generation. (C) Shown here is a typical complex that includes some spherical HBsAg particles (diameter, 22nm) (A) and a few Dane-like particles (42nm) (B) (magnification 14,500×; bar: 200nm; accelerating voltage: 160kV).

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Samples containing medium of the HepG2.RL1 cells were visualized by ITEM analyses. Immune complexes were obtained from samples that had been immunoprecipitated with antiserum directed against HBsAg. Some of these immune complexes had several Dane-like particles of size ∼42nm and a few spherical HBsAg particles (∼22nm) (Fig. 4C). However, when samples containing medium of HepG2 cells were examined, virus-associated particles were not observed.

Upon exposure to various concentrations of NCC for six days, the results from the MTT assay showed that NCC inhibited the growth of HepG2.2.15 cells and HepG2.RL1 cells with CC50 of 725.81±23.34μM and 782.38±12.17μM, respectively, indicating almost the same extent of toxicity on the two cell lines, whereas CC50 for adefovir was 259.81±13.49μM and 290.57±21.31μM in HepG2.2.15 cells and HepG2.RL1 cells, respectively.

The HBsAg level from HepG2.RL1 was very low even in the drug-free group (data not shown) and hence the evaluation was based only on HBeAg. The dose-response relationship showed that lamivudine did not inhibit HBeAg secretion at the highest concentration (100μM) in HepG2.RL1 cells, whereas wild-type HBV showed considerable inhibition (Fig. 5A). However, adefovir treatment dose-dependently reduced the secretion of HBeAg in wild-type and lamivudine-resistant HBV (Fig. 5B).

Fig. 5.

Drugs susceptibility to antigens secretion and viral replication.

HepG2.2.15 cells and HepG2.RL1 cells were treated with lamivudine and adefovir for six days. Cell-culture supernatants were assayed using a specific ELISA kit. Viral DNA was extracted from the culture medium and cells, and then quantified by qPCR using a commercial kit. Experiments were done from wild-type and lamivudine-resistant HBV isolates, on the inhibitory effects of lamivudine (A) and adefovir (B) on HBeAg production; inhibitory efficiency of lamivudine (C) and adefovir (D) on extracellular production of HBV DNA; inhibitory efficiency of lamivudine (E) and adefovir (F) on intracellular production of HBV DNA. Mean values of inhibition from three independent measurements are shown as bars with standard-error values indicated at the top. **p<0.01, *p<0.05, compared with the no-drug control group. Black bar: wild-type HBV; gray bar: lamivudine-resistant HBV with rtL180M/M204V mutations.

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Viral DNA was extracted and subjected to qPCR analyses at the end of treatment. In terms of inhibitory potency on extracellular DNA replication, EC50 of lamivudine from wild-type HBV and HepG2.RL1 cells were 0.16±0.06μM and >100μM, respectively (Fig. 5C), which indicates a >625-fold-resistance. Based on the dose-response histogram, adefovir inhibited the replication of mutant HBV almost as efficiently as it did with wild-type HBV, with EC50 of 4.18±0.18μM and 3.69±0.50μM, respectively (Fig. 5D). With respect to intracellular DNA, lamivudine was not active on the rtL180M/M204V double mutation carrying HepG2.RL1 cells (EC50>100μM) compared to the wild-type HBV strain (EC50=0.018±0.01μM), which represents a decreased susceptibility of more than 5556-fold (Fig. 5E). Adefovir inhibited replication of intracellular DNA in HepG2.RL1 cells, consistent with that seen in wild-type HBV, with EC50 of 2.24±0.11μM and 1.36±0.08μM, respectively, both in a dose-dependent manner (Fig. 5F).

Inhibitory efficiency of antiviral agents was assessed only with HBeAg. NCC dose-dependently inhibited HBeAg production by HepG2.2.15 cells and HepG2.RL1 cells (Fig. 6A). Consistent with the inhibitory effect on HBeAg production, NCC inhibited the replication of wild-type HBV and resistant HBV intracellular DNA in a dose-dependent manner. Importantly, there had no significant difference of the inhibitory rate between HepG2.2.15 cells and HepG2.RL1 cells (Fig. 6B), and EC50s for the inhibition of replication of intracellular HBV DNA by NCC were 2.23±0.79μM and 0.84±0.12μM, respectively. Moreover, NCC exposure produced dose-dependent inhibition of the replication of extracellular HBV DNA (Fig. 6C), with EC50s of 0.14±0.07μM and 0.46±0.09μM, respectively, for the HepG2.2.15 cells and HepG2.RL1 cells.

Fig. 6.

Effect of NCC on antigen secretion and viral replication.

HepG2.2.15 cells and HepG2.RL1 cells were treated with the indicated concentrations of NCC for 6 days. HBeAg was tested using a specific ELISA kit (A). The intracellular DNA (B) and extracellular DNA (C) was quantified by qPCR using a commercial kit. Experiments on the inhibitory effects of NCC on HBeAg production and replication of HBV DNA from HepG2.2.15 cells (black bar) and HepG2.RL1 cells (gray bar) were performed in triplicate. Mean values of inhibition from three independent measurements are shown as bars with standard-error values indicated at the top. **p<0.01, *p<0.05, compared with the no-drug control group.

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