| HBV, the prototype member of the family Hepadnaviridae, has the high species and tissue specificity: it infects only humans and humanoid primates or cultured primary hepatocytes of these hosts. Besides virus uptake, viral promoters and enhancers confer hepatocyte specificity during replication. For studying hepatitis B virus infection, no permissive cell line or small animal is available. Stable transfected cell lines and transgenic mice which contain hepadnavirus genomes produce virus, but, unlike in natural infection, from an integrated viral transcription template. Adenovirus transfers genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. The adenovirus-mediated genome transfer efficiently initiated hepadnavirus replication from an extrachromosomal template in the target cells. To transfer hepadnavirus genomes across the species barrier, we used the recombinant adenovirus containing the whole HBV genome, infected L02 cell lines and KM mice as a new cell and animal model. With this cell line and animal, we studied the influence of IFNα-2a and Peg-IFNα-2a on the HBV replication in vitro and in vivo, discussed the difference to the HBV cccDNA after treated with the IFNα-2a and Peg-IFNα-2a.1. Biological characteristics of the recombinant adenovirus.We used the recombinant adenovirus which containing the whole HBV genome to infect the cells, observed the expression of the antigens, and introduced the biological characteristics of the recombinant adenovirus.We used the recombinant adenovirus to infect the 293 cells to generate viruses. By using the PCR technique, the integration of HBV DNA into the adenovirus genome was identified, and the viral genomes were stable during the passage in 293 cells. The expressions of HBV antigens were detected in culture medium of L02 cells infected by the recombinant adenovirus. When L02 cells were infected at various multiplicity of infection (MOI), the expressions of HBsAg and HBeAg increased with MOI. The cells were tended to aging and dropped off when the MOI is higher than 50.One chicken hepatoma cell and two types of human hepatocytes were infected by the recombinant adenovirus at 25 pfu / cell. HBV specific mRNA in these three cells infected by recombinant adenovirus was detected by RT-PCR. HBsAg and HBeAg were detected in the culture medium by ELISA. It showed that the HBV could replicate in the heterogeneous hepatocytes, and the HBV infectious state could be established in these cells after infected by recombinant adenovirus, it indicated that there was no strict species restriction to HBV replication, and the HBV replication was dependent on the hepatocytes. The expression of HBV antigens were increased by the time and reached the maximum at the day 5 or day 6. The antigens in human hepatocytes were higher than in chick hepatoma cells, it should be the tissue specificity of the antigens. The antigens in HepG2 cells were higher than in L02 cells, it seemed that the tumorous cells have the higher metabolism than the normal cells.The adenovirus genome can be detected in nucleus after infection, and after 3 days in culture medium. It indicated the high ability of genome transfer of the recombinant adenovirus, and the cells were sensitive to the recombinant adenovirus. 2. Dynamic analysis of the HBV replication in L02 cell infected by the recombinant adenovirus.The L02 cells were infected with the recombinant adenovirus and the cells were able to express the HBV antigens. The HBsAg and HBeAg were detected by ELISA, HBV DNA by real time PCR and HBV cccDNA with the special primers. Then the replication of HBV could be illuminated in L02 cells.The L02 cells were infected by the recombinant adenovirus at 25 pfu / cell, washed 3 times with PBS 3 hours after the virus adsorbed, applied with 1ml of DMEM with 10% FBS and then incubated overnight. The cells and the culture medium were collected at 24 hours after virus infected, 8 days continuously, and saved at -20℃.The DNA of the culture medium was extracted by using a blood DNA extract kit, and detected the HBV DNA by real time PCR.The intracellular HBV cccDNA was extracted with non-proteinase lysis buffer, the cccDNA and rcDNA were distinguished by the different binding ability to the protein, the linear dsDNA and nicked DNA were eliminated by treating with Mung Bean nuclease, and then cccDNA was detected by real-time PCR with the special primers.The HBV cccDNA were detected with special primers from 2 patients' sera containing 107 and 106 copies / ml of HBV DNA, respectively. The result of negative control showed that the primers were specific to HBV cccDNA.The plasmid was diluted into 102, 103, 104, 105, 106, 107 copies / ml to detect the primers' sensitivity. The plasmids of 103-107 copies / ml were positive, so the sensitivity of this method was at the level of 103 copies / ml.HBV cccDNA was detectable 1 day after infected by recombinant adenovirus, reached the maximum at the 4th day, then descended after that point, but still maintained a stable level. It seemed that the stability of the cccDNA could satisfy the virus replication but was innocuous to the cells, which was in favor of the cells' long-term infectious state.The HBV DNA in the culture medium was detected by real time PCR, and it had the highest content at the 5th day.The HBsAg and HBeAg in the culture medium were detected by ELISA, both increased with the time, and reached the maximum at the 6th day, during this period the cells were in a stable state. With the cells aging with the time and their metabolism decreasing, the virus expression and replication depended on the cells were decreased.The HBV cccDNA, HBV DNA, HBsAg and HBeAg were analyzed by Spearman correlation. The results showed that the HBV DNA in culture medium was correlated with HBsAg and HBeAg (rs=0.881, P=0.004; rs=0.857, P=0.007), but there was no correlation between the HBV cccDNA and the antigens (P＞0.05).3. Influence of Peg-IFNα-2a and IFNα-2a on the HBV replication in vitro.With the L02 cells infected by the recombinant adenovirus, the difference of the HBV DNA in culture medium, HBV cccDNA in nucleus, and the change of HBsAg and HBeAg fore-and-aft treatment with the Peg-IFNα-2a and IFNα-2a were observed, the antivirus ability of Peg-IFNα-2a and IFNα-2a in vitro was compared, and the influence of the two IFN on the cccDNA, the first intermediate of HBV discussed.L02 cells were infected and treated as shown above. Then the cells were divided into 3 groups and each group tripled, one group was treated with the IFNα-2a (with the consentration gradients as 100 IU/ml, 500 IU/ml, 1000 IU/ml, respectively), one with the Peg-IFNα-2a (3.6×10-3μg/ml, 1.8×10-2μg/ml, 3.6×10-2μg/ml), and the another was as control. All the cell cultures were maintained at least for 7 days. Cytotoxic assay showed the Peg-IFNα-2a and IFNα-2a were innoxious to the cells in these three dosages (P＞0.05).Seven days after the L02 cells treated with the medicines, the culture media were collected, the cells after washed 3 times and digested with trypsin, were also collected. The DNA in culture medium was extracted with a blood DNA extraction kit, and the cccDNA with the non-proteinase lysis buffer.In comparison of HBsAg and HBeAg between the study and control groups, all 3 dosages of the Peg-IFNα-2a group and 100 IU/ml of IFNα-2a group were not significantly different (P＞0.05), while 500 and 1000 IU/ml of the IFNα-2a group had significant difference (P＜0.05, P＜0.001). It showed that the IFNα-2a could decrease the expression of the HBV antigen in L02 cells after infected by the recombinant adenovirus at the dosages more than 500 IU/ml.The HBV DNAs in medium of the Peg-IFNα-2a group with all three dosages were not significantly different compared with the control group (P＞0.05). While those of IFNα-2a group with both 500 and 1000 IU/ml dosages were significantly different (P＜0.05). It indicated the IFNα-2a could suppress the HBV replication in medium at the dosages≥500 IU/ml.Analyzed for the HBV cccDNA in nucleus, it showed no significant difference of all groups (P＞0.05). It indicated that the medicines were no obvious effect on the HBV cccDNA.4. Influence of Peg-IFNα-2a and IFNα-2a on the HBV replication in vivo.In the animal model, the antivirus ability of the IFNα-2a and Peg-IFNα-2a were compared in vivo, and the influence of the two IFNs on HBV cccDNA was discussed.Three groups of 4 weeks old KM mice were infected by a single injection of 2×109 pfu of purified recombinant adenovirus into the tail vein, one group was treated with IFNα-2a (1.7×103 IU, thrice a week), one group with Peg-IFNα-2a (0.06μg, once a week), and another group with saline as control by subcutaneous injection, respectively. Samples of mice serum and livers were taken and tested for HBV replication at the 4th weeks and the 8th weeks.The mice sera DNA and livers DNA were extracted and the HBV DNA was detected with HBV special primers by PCR, and all of the results were positive.The adenovirus genome was detected in mouse sera and livers with adenovirus specific primers by PCR, the result was positive in the mice livers infected at the 4th week, but it was negative in those infected at the 8th week. All mice sera were negative for adenovirus DNA. It indicated that there wasn't the recombinant adenovirus in mice sera, the HBV DNA in sera were descended from the virus replication.The RNA of the infected mice was extracted at the 4th week and the 8th week, treated with 0.5μl Dnase I to remove the contaminated DNA, and then the HBV RNA was detected by RT-PCR. The special HBV RNA was detected in all of the mouse livers, it showed that there were HBV replicating intermediate in mouse livers, so the HBV infected mouse was established by the recombinant adenovirus in vivo.The IFN-γin mouse sera was detected by ELISA, of the medicine groups, it was significantly different compared with the control (P＜0.05), but the IFN-γin the 4th week and in the 8th week had no significant difference (P＞0.05). The IFN-γof the medicine groups were higher than the control (P＜0.05), and the Peg-IFNα-2a group was higher than the IFNα-2a group (P＜0.05), too. This indicated that both the Peg-IFNα-2a and IFNα-2a had the immune-regulation ability in vivo, but the immune-regulation ability of Peg-IFNα-2a was higher than IFNα-2a.Quantitatively assay of HBV DNA in mice sera showed that the viral load at the 4th week and the 8th week between three groups were not significantly different (P＞0.05). In comparison of two study groups with the control, the HBV DNA levels were distinctly declined (P＜0.05). It indicated that the Peg-IFNα-2a and IFNα-2a could suppress the virus replication in vivo. While the HBV DNA of the Peg-IFNα-2a group to the IFNα-2a group was compared, there was no significant difference (P＞0.05), we considered that the Peg-IFNα-2a and IFNα-2a had the same effect on the HBV DNA in mice sera.With quantitatively analysis of the HBV cccDNA in mice livers, it showed that in three groups, the cccDNA in the 4th week and 8th week were not significantly different (P＞0.05). This indicated that the IFNα-2a and Peg-IFNα-2a have no obvious effect on the liver cccDNA.Conclusion: The recombinant adenovirus mediated HBV genome is transferred into culture cells in vitro and into mice in vivo, the HBV antigens can be detected in the culture medium, and the HBV replication intermediates can be detected in the cells and in the mice. The antivirus ability of the IFNα-2a is higher than the Peg-IFNα-2a in vitro, but the Peg-IFNα-2a has higher immune-regulation ability than IFNα-2a in vivo. In the short term, both of them can suppress the HBV replication in sera, but have no obvious effect on the HBV cccDNA.
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