Patients
A total number of 106 patients with chronic HBV infection who visited our hospital from January 2010 to December 2015 were enrolled in the study. They were treated with NAs for more than one year, and scheduled follow-up every 3-6 month. They exhibited viral breakthrough during antiviral treatment and were identified rtA181T/sW172* or rtA181V mutations. The diagnostic criteria were according to the Chinese guidelines for prevention and treatment of CHB (2015 version) [13]. This study was approved by the institutional review board of China Nanjing bayi hospital and all aspects of the study comply with the Declaration of Helsinki. The Ethics Committee specifically approved that no informed consent was required because this was a retrospective study and the data were analyzed anonymously.
Laboratory tests
Biochemical Indicators were measured by Olympus AU5400 Chemistry System (Beckman Coulter, Switzerland). HBV markers, including hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and anti-HBe, were tested by Chemiluminescent Assay (Architect i2000SR, Abbott Diagnostics, Abbott Park, IL, USA). HBV DNA was quantified by real-time PCR using a commercial detection kit (Kehua Bio-Engineering, China) with the aid of Light Cycler Detection System (ABI 7300 Realtime PCR system, USA). The lower limit of detection for this assay is 50 IU/mL. Serum HBV DNA was extracted using the QIAamp blood mini kit (Qiagen, Germany). The fragments (nt130–nt1129), encompassing the RT domain, were amplified by nested PCR and the HBV strains were detected by direct Sanger sequencing as previously described [14]. Substitutions at positions rt80, rt173, rt180, rt181, rt184, rt202, rt204, rt236, and rt250 were taken as resistance mutations for analysis. HBV genotypes classification assignment were based on phylogenetic analysis of the 1225-bp-long S/Pol gene sequence.
Clone sequencing analysis
Serum samples from 15 patients with rtA181T/sW172* mutation were randomly selected for clone sequencing analysis. Of them, 5 samples were genotype B and 10 samples were genotype C.The PCR product was purified and recovered according to the instructions in the PCR Purification Kit (Axygen, Hangzhou, China), connected to T-A vector at 4℃ overnight, then transformed into E.coli DH5α competent cells. More than 18 positive clones were selected for sequencing from each plate. The sequences were aligned using the Clustal W program in Megalign version 3.17 software.
Construction of 1.24-fold genome HBV vectors
Ae-JPN, Ba-JPN58, C-JPNAT and D-US68 HBV plasmid kindly provided by Sugiyama[15] were used as wild type templates to construct rtA181T/sW172* mutations strain, respectively. Site-directed mutagenesis (Stratagene, La Jolla, CA) were carried out to introduce these mutations. The sequence of the mutagenesis primers for different genotypes are shown in Table 1.
Table 1 Sequences and location of primers for construct HBV rtA181T/V mutation
Primer set
|
Primer name
|
Sequence(5’-3’)
|
Position(nt)
|
Sense
|
AT-F
|
5′-GTCCGTTTCTCTTGACTCAGTTTACTAGTGCC-3′
|
656-687
|
Antisense
|
AT-R
|
5′-GGCACTAGTAAACTGAGTCAAGAGAAACGGAC-3′
|
687-656
|
Sense
|
CT-F
|
5′-GTCCGTTTCTCCTGACTCAGTTTACTAGTGCC-3′
|
656-687
|
Antisense
|
CT-R
|
5′-GGCACTAGTAAACTGAGTCAGGAGAAACGGAC-3′
|
687-656
|
Sense
|
DT-F
|
5′-GCCCGTTTCTCCTGACTCAGTTTACTAGTGCC-3′
|
656-687
|
Antisense
|
DT-R
|
5′-GGCACTAGTAAACTGAGTCAGGAGAAACGGGC-3′
|
687-656
|
The primers were designed completely complementary, the bases marked were site-directed mutagenesis. The primers for genotype B and genotype A are the same.
Cell transfection, Relative replication and HBsAg expression yield assays
HepG2 human hepatoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS). After 24h of culture, cells were transiently transfected with 5 μg plasmids using FuGENE HD Transfection (Roche, Switzerland). Transfection efficiency was measured by cotransfection with 0.1 μg pSEAp and determination of alkaline phosphatase activity in cell supernatants using SEAP Reporter Assay Kit (Roche, Switzerland).
After 72h post-transfection, HBsAg in cell culture supernatants were detected by ELISA, HBV virions from supernatants were quantified by real-time PCR after the following process. 20µl of 10×Reaction Buffer (100mM Tris[pH7.5], 25mM MgCl2, 1mM CaCl2), 2µl of 20mg/ml DNAse I, and 1µl of 10mg/ml RNAse A (Sigma, Germany) were added to the 180µl solution and incubation at 37℃ for 2 h, followed by inactivation reaction at 70℃ for 10 min with EDTA at a final concentration of 10mM [16]. The HBV replication and HBsAg expression yield were standardized relative to wild type genotype A, defined as 1.0. All experiments were performed 3 times.
Statistical analysis
Quantitative data were expressed as means ± standard deviations and analyzed by independent samples t test or U test. Count data constitute were used χ2 test. Statistical analysis was carried out with SPSS 13.0 software. Significance was set at P < 0.05.