Animal experiments, sample collections and statistics
Animal experiments were carried out in accordance with the guidelines for care and use of laboratory animals issued by Chinese government. Animals were housed at the Laboratory Animal Center of Guangxi Medical University under monitoring of veterinarians. Details have been reported previously
The founders of the tree shrews used in this study were derived from a population of wild tree shrews (Tupaia belangeri chinensis) originating from the Kunming Institute of Zoology, Chinese Academy of Science (Yunnan, China). These animals were genetically inhomogeneous and continued to be bred in our laboratory for years. Newborn descendants from this population were utilized for experiments.
Newborn tree shrews were injected with 0.3 ml HBV inoculum twice subcutaneously, on the first and third day after birth, respectively. Six weeks after inoculation, blood samples were collected once every 4–6 weeks, liver samples were collected every 6–12 weeks using anesthesia (1% pentobarbital and ketamine hydrochloride). Animals were observed for at least 24 weeks after inoculation, or observed continuously as long as HBV-infection marker could be detected. A portion of each serum sample was tested immediately for HBV immunological markers, including HBV surface antigen (HBsAg), HBV surface antibody (HBsAb), HBV e antigen (HBeAg), HBV e antibody (HBeAb) and HBV core antibody (HBcAb) by enzyme linked immunosorbent assay (ELISA), or further by time-resolved immunofluorescence analysis (TRFIA). The other portion of serum sample was stored at −80°C for later molecular tests. A piece of each liver biopsy sample was frozen immediately by immersion in liquid nitrogen followed by storing at −80°C, and the remaining was fixed within 10% (v/v) neutral formalin and then paraffin embedded.
Infection rates of the two inocula, i.e. serum collected from human HBV-carriers and serum from a HBV-infected tree shrew, were analyzed by Chi-Square test with SPSS 13.0 statistics software (IBM, Chicago, USA).
Inocula and positive controls
Inocula for injecting the experimental tree shrews included serum collected from human HBV-carriers and serum from an infected tree shrew. The HBV DNA copy number was ≥107/ml in human serum and 104–105 /ml in tree shrew serum, which was validated prior injection.
Positive control samples included human sera from chronically HBV-infected patients, and liver tissues obtained from HBV-infected patients who had undergone surgical resections for hepatocellular carcinoma (HCC) at Guangxi Tumor Hospital of China.
The study protocol was approved by the Ethical Committee of Guangxi Tumor Hospital in accordance with the guidelines issued by Chinese government, which conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Informed consents in writing were obtained from all participating patients.
Detection of HBV immunological markers in serum by ELISA and TRFIA
Serum specimens of the experimental tree shrews were analyzed qualitatively by ELISA, for HBsAg, HBsAb, HBeAg, HBeAb and HBcAb. HBsAg positive specimens were further analyzed quantitatively by TRFIA.
Both tests of ELISA and TRFIA were carried out by the Clinical Laboratory Center of Guangxi Tumor Hospital (Nanning, China), using validated procedures for their clinical specimens. Kehua Bio-engineering (Shanghai, China) was the manufacturer for the ELISA kit, which was operated with an automatic processor (ML-FAME, AusBio Company, Bonaduz, Switzerland). TRFIA was conducted also by using a commercial kit (Xinbo Biotechnology, Suzhou, China) and an automated immunoassay system (Wallac AutoDELFIA, PerkinElmer Company, Turku, Finland).
Detection of HBV DNA in serum and liver by fluorescence quantitative polymerase chain reaction (FQ-PCR)
Recovery and extraction of viral DNA from serum was performed according to the manufacturer’s instruction provided by a FQ-PCR kit (Kehua Bio-engineering Co. Shanghai, China) and a previously published procedure
. Total DNA extraction and HBV DNA detection from liver tissue were performed following the procedures described by Cacciola et al.
. Briefly, liver tissue was homogenized in digestion buffer that contains NaCl, Tris–HCl, sodium dodecyl sulphate and ethylenediaminetetraacetic acid, followed by digestion with proteinase K overnight, and extraction with phenol/chloroform/Isoamyl alcohol (25:24:1). The concentration of liver total DNA was determined using a spectrophotometer at 260 nm.
FQ-PCR amplification and analysis was carried out by the Clinical Laboratory Center of the First Hospital of Guangxi Medical University (Nanning, China), utilizing the same standard procedures that were implemented for their clinical samples. The amplification and analysis was performed on an ABI 7300 analyzer (Applied Biosystems, Foster City, CA, USA). The amplification conditions were 50°C 2 min, 94°C 2 min, followed by 40 cycles of 94°C 10 sec and 60°C 30 sec. Each round of amplification was accompanied by a calibration test with a serial of standards supplied within the FQ-PCR kit.
Per serum-derived HBV DNA was determined as copy/ml. Liver tissue-derived HBV DNA was calculated as copy/μg liver DNA
. According to the kit’s manufacturer, the threshold for determining positivity of serum samples had been as ≥103 copies/ml. However, since no manufacturer’s critical threshold value had been recommended for liver tissue, we determined it as ≥104 copies/μg liver DNA. This assumption was based on a series of limiting dilution experiments using positive and negative controls (data not shown).
Detection of HBV DNA in liver by Southern blot
Southern blot analysis was performed in the laboratory of Professor Blum at Freiburg University of Germany, using the procedures previously described by his group
. Briefly, 20 μg of digested tree shrew liver DNA was separated on a 1.4% agarose gel, then transferred onto nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Hybridization was performed in Roti-Hybri-Quick solution (Roth Chemikalien, Karlsruhe, Germany) using a 32P-labeled full-length sequence of wild-type HBV DNA probe.
Detection of HBV cccDNA in liver by nested polymerase chain reaction (nPCR)
The detection was performed according to the protocol published by Singh et al.
. Briefly, DNA extracted from tree shrew liver tissue was digested with Plasmid-SafeTM ATP-Dependent DNase (Epicentre Technologies, Madison, USA) to reduce the non-ccc HBV-DNA. DNA purification was then performed using a kit from Tiangen Biotech Co. (Beijing, China). Primers for amplification were synthesized by Sangon Biotech Co. (Shanghai, China). The sequences of outer primers were 5’-GCTTTGCTGCTCCATTTACAC-3’ (nt1013-nt1033) and 5’-TTCCGGAAGTGTTGATAAGAT–3’ (nt2335-nt2315), of inner primers were 5’-CCGACCACGGGTCGCACCTCTC-3’ (nt1513-nt1534) and 5’-CTTGAACAGTAGGACATGAACA-3’ (nt1847-nt1868). Cycling conditions for the initial amplification of nPCR consisted of 3 min pre-denaturation at 94°C; 30 sec denaturation at 94°C, 1 min annealing at 50°C and 2 min extension at 72°C for 35 cycles, and 7 min final extension at 72°C. The conditions for the second amplification consisted of 3 min pre-denaturation at 94°C; 20 sec denaturation at 94°C, 20 sec annealing at 50°C and 20 sec extension at 72°C for 35 cycles; and 7 min final extension at 72°C. The recombinant plasmid pUC18-HBV and sterile water were used as positive and negative controls, respectively. The amplification product was electrophoresed in 1.7% agarose gel. The 356 bp DNA band was purified and the sequence was confirmed by a commercial vendor (SinoGenoMax Co. Beijing, China).
HBV-genotype analysis by nPCR
Analyses of HBV genotypes A through F were performed on the human sera which had been used as inocula and the sera obtained from infected tree shrews. The procedure as well as the sequences of genotype-specific primers have been previously published by Natio et al.
. The primers were synthesized by Sangon Biotech Co. (Shanghai, China). Briefly, the first round of PCR amplification employed universal outer primers for all of the six HBV genotypes, the products of this reaction were then used as template for the second round of PCR utilizing two different mixtures (A and B) of inner primers. Mix A consisted of a sense primer common for genotypes A, B and C, and three different antisense primers specific for genotypes A, B and C, respectively. Mix B consisted of an antisense primer common for genotypes D, E and F, and three different sense primers specific for genotypes D, E and F, respectively. The final products were subjected to gel electrophoresis.
Detection of HBsAg and HBcAg by immunohistochemistry and histopathological examination on liver biopsies
Presences of HBsAg and HBcAg in paraffin-embedded liver biopsies were examined by Immunohistochemistry assays. Monoclonal antibodies of mouse anti-human HBsAg (Zhongshan Goldenbridge Biotech, Beijing, China) and mouse anti-human HBcAg (Maxim Bio, Fuzhou, China) were used in conjunction with UltraSensitiveTM S-P staining kit and DAB kit (Maixin Bio, Fuzhou, China), following the instructions provided by manufacturer. As positive control, HCC-surrounding liver tissue from HBV-infected HCC-patients was used.
Hematoxylin and eosin (HE)-stained liver biopsies were analyzed and interpreted by certified pathologists.
Study specific criteria defining “confirmed” and “suspected” chronic-infection
For this study, we established the criterion for “confirmed chronic-infection” for which the same animal had to sustain detectable serum HBsAg in addition to either serum or liver detectable HBV DNA in excess of 48 weeks post inoculation. The criterion for “suspected chronic-infection” was the requirement for detectable HBV DNA over at least 48 weeks post inoculation, on at least two or more occasions. For “suspected chronic infection”, the HBsAb detection remained negative or was only occasionally positive during the early period following inoculation.