Patients and blood samples
Blood samples were provided by You’an Hospital in Beijing. This study was approved by the Institutional Review Board of the Beijing Institute of Genomics and the Ethics Committee of Beijing You’an Hospital of Capital Medical University. Informed consent was obtained from all patients. Patients were diagnosed as chronic carrier (CC), chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) according to the guidelines on the prevention and treatment of chronic hepatitis B in China (2010)
. No patients had co-infections with HCV, HDV, or HIV. Blood samples of 5ml were collected, cells and sera were then separated and stored at −20°C. From the few hundred stored samples, we successfully amplified and sequenced 51 whole genomes from 51 individuals. Additionally, preS clone sequencing was performed in another cohort of 52 patients for fine mapping of deletion substructure.
DNA quantification and HBV serological marker detection
Viral DNA titers were quantified using the FQ-PCR Kit for HBV (DaAn Gene Co., Guangdong, China) on a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems, CA, USA). Serological markers were determined by an Electrochemiluminescence Immunoassay on a Roche E170 Modular Immunoassay Analyzer (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s protocol.
Viral DNA extraction and amplification
Viral DNA was extracted from 400 μl sera per sample using the QIAamp MinElute Virus Spin (Qiagen, Hilden, Germany). All DNA samples were stored at −20°C.
Whole genome amplification was performed using LA Taq (Takara, Osaka, Japan) according to the method described by Günther et al., with the primers for P1(1821 to 1841), CCGGAAAGCTTGAGCTCTTCTTTTTCACCTCTGCCTAATCA, and P2 (1823 to 1806), CCGGAAAGCTTGAGCTCTTCAAAAAGTTGCATGGTGCTGG
. The lowest DNA amount required for amplification was 103 copies/ml in our experimental system. Sequencing primers are listed in Additional file
1: Table S1, and the primers SP5 and SP9 were also used for preS region amplification. Hot start PCR for the preS region was performed with the following cycle: 95°C for 2 minutes and 30 seconds, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 90 seconds, and elongation at 72°C for 3 minutes. All reactions were performed on a PTC-200 Peltier Thermal Cycler (MJ Research, MA, USA).
Viral DNA sequencing
After purification via the Montage PCR96 column (Millipore, MA, USA), PCR products were sequenced on a Prism 3730 (ABI, USA). Contigs were assembled using SeqMan (DNAstar 5.0, WI, USA), and sequences were aligned using ClustalW for further analysis. All mutations were checked manually. Whole genomes mentioned in this study are defined as >97% of full length and sequencing gaps at the end of the genome have no overlaps with deletion hotspots.
The boundaries of deletion regions that appeared in the sequencing electropherogram were determined by reading from both directions. The regions of interest were amplified by PCR and the products were cloned into a pMD18 T vector (Takara, Osaka, Japan) followed by sequencing of 5–10 positive clones per sample. NCBI accession numbers for all sequences are listed in Additional file
1: Table S2.
Construction of HBV mutants and examination of their antiviral resistance
Candidate deletions were introduced into the HBV-expression plasmid Yi026-pcDNA3.1/Zeo(−) using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, CA, USA). The plasmid, harboring a 1.1X overlength genome of HBV (ayw), was kindly provided by Yi Ni and Stephan Urban from the University of Heidelberg (Heidelberg, Germany). Introduced mutations were verified by plasmid re-sequencing.
HuH7 cells were seeded into 10 cm2 dishes at 1.5 × 106 cells/dish, reaching around 90% confluency before transfection the following day. Cells were transfected using 24 μl FuGENE®HD (Roche, IN, USA)) reagent with 8 μg of plasmid DNA. 16–20 h post-transfection, transfected cells were washed twice and then seeded into a 96-well plate at 3 × 104 cells/well. The cells were treated with serial dilutions of four drugs in fresh medium for 3 days, including lamivudine (LMV), adefovir (ADV), entecavir and tenofovir (Sequoia Research Products Limited, UK). The supernatants were collected after centrifugation at 1500 × g for 5 min, and then prepared for HBV DNA extraction using the BioSprint 96 One-For-All Vet Kit (Qiagen, Hilden, Germany). Half maximal inhibitory concentrations (IC50) were calculated for each construct where the resistance factor is calculated as the IC50 of mutant divided by the IC50 of the wt strain. The amount of HBsAg produced by each strain was determined by the AxSYM HBsAg assay (Abbott Laboratories, IL, USA).
SPSS 13.0 was used for logistic regression analysis, t-tests and Fisher exact tests (FET).