In the present study, an HBV genotype B HI mouse model was established. After HI with pAAV-HBV1.3B, HBsAg and HBV DNA were detected in peripheral blood. Southern blot and immunohistochemical staining analysis revealed that HBV DNA and HBcAg were detected in the liver tissue of HBsAg-positive mice. Different hepatitis B virus genotypes may have distinct virologic characteristics, which may correlate with antiviral curative effects and clinical outcome [29, 30]. The majority of HBV infections are distributed in the Asia-Pacific region. In these regions, patients are mainly infected with genotypes B and C. Previous studies showed that in China and Taiwan, approximately 90% of patients under thirty-five years old who are infected with HBV would develop HCC and most of them carried an HBV genotype B infection [30, 31]. Now, we have a system at hand to examine the replication of HBV genotype B clone in the mouse models.
In present study, we investigated the effect of the HBV genome length on HBV replication competence to find the optimal structure of replication competent HBV genotype B clone. Three clones with 1.1, 1.2 and 1.3 fold over length HBV genotype B genomic DNA were constructed based on the pBluescript II KS (+) vector. Schematic representation of the 1.1, 1.2 and 1.3 fold HBV genome used in this study are shown in Additional file 1: Figure S1B. The plasmid with 1.3 fold genomes containing a complete extra copy of the entire EnhI, Enh II and HBx gene as shown in Additional file 1: Figure S1B which is a suitable HBV construct for transfection and HI experiments. When the cloned plasmids were transfected into Huh-7 cells or hydrodynamically injected into BALB/c mice, pBS-HBV1.3B demonstrated significantly high-level replication and gene expression in comparison to pBS-HBV1.1B and pBS-HBV1.2B (Figure 1). Increasing the length of the 1.3 fold over length HBV genome did not further improve the replication competence. Nevertheless, the replication competence declined and could not reach the original level of HBV in nature infection state when the length is less than 1.2 fold of the HBV genome. Durantel et al. and other groups used 1.1 fold over length HBV genome for studies on drug resistant HBV isolates [32, 33]. However, they added strong foreign promoters to enhance the HBV transcription and replication. In conclusion, the 1.3 fold HBV genome length containing a complete extra copy of the entire EnhI, EnhII and HBx gene as described in the present study could be regarded as an optimal length for HBV replication constructs with the authentic HBV sequence, independent on a foreign promoter.
To establish a persistent replication HBV mouse model with the HBV genotype B clone, 1.3 fold over length HBV genotype B genome was sub-cloned into the pAAV-MCS vector and introduced into C57BL/6 mice by HI. Huang et al. (2006) found that the AAV vector contributed to the persistence of HBV in mouse liver. Therefore, we used a similar vector pAAV-MCS, to construct HBV genotype B replicational clone in this study. This vector also has the ITR (inverted terminal repeats) structure, which was considered as a key factor for its stable existence in vivo by integration or non-integration way. Because the vector does not express other proteins except that encoded by the target genes, the vector backbone has no direct effect on the immune system.
After HI, the mice presented higher HBV DNA level, but a lower positive rate for HBsAg in serum compared with pAAV-HBV1.2 injected mice . Apparently, HBV replication is not strictly correlated with HBsAg production, as known for patients [34–38]. The result indicated that the model is more robust when pAAV-HBV1.2 genotype A is used as compared with pAAV-HBV1.3B. Huang et al. (2006) confirmed that the tolerance toward HBV surface antigen in this model was due to an insufficient cellular immunity against hepatitis B core antigen, as documented in humans. We repeated some experiments and obtained the same results (Figure 4A and B). The difference in plasmid backbones between both clones needs to be investigated further.
The antigenemia and production of HBV particles were highly reproducible in the persistent HBV replication mouse model based on HI making this model appropriate for the evaluation of the efficacy of anti-viral drugs. Therefore, we further investigated the anti-viral effect of vector-expressed HBx siRNA in this model. Co-application of the HBx siRNA expression plasmids treatment of these mice induced a significant inhibition of HBV replication and expression. When pAAV-HBV1.3B was co-injected into the tail veins of C57BL/6 mice with either pSB-HBxi285 or pSR-HBxi285, HBV expression and replication were efficiently inhibited. There are two methods commonly used to silence target genes. Chemical synthesized siRNAs have a good specificity and are commonly used on cells by transfection. However, such siRNAs, if not chemically modified, are instable and eliminated rapidly in vitro and in vivo. The production of siRNAs, particularly with specific chemical modifications, is very expensive. Another approach is to use siRNA transcription vectors. The disadvantages to use such as retroviral vectors are, for example, the lower transfection efficiency and slow action compared with chemical synthesized siRNA. However, the vector-based approach has some advantages regarding the stability and costs. It is important to prolong the expression of siRNAs to maintain the inhibition of HBV. Moreover, when the pSuper.Retro vector was transfected into a packaging cell line such as 293 T cell line, it could produce retroviral supernatants and obtain a higher rate of stable cell integration (The Manual of pSuper RNAi system, OligoEngine, Washington, USA) [39, 40].
HI of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. The early reports suggested that a large portion of hepatocytes could be reached with this technique [41–44]. Therefore, it could be a useful method to test the efficacy of RNAi based antiviral therapies in the mouse model. Certainly, there is still a challenge how to transfer the knowledge gained in this system into human situation.
In this study, non-viral (pSuper.Basic) and retroviral vectors (pSuper.Retro) were used to construct shRNA (small hairpin RNA) expression plasmids  targeting the HBx gene of HBV. The pSuper.Retro vector contains the H1 promoter which is a RNA polymerase III like promoter and can transcript shRNA steadily, and showed persistent inhibitory effect in vitro[46, 47]. The pSuper.Retro vector includes 3′ and 5′ terminal LTR structure of murine stem cell virus. The pSuper.Retro vectors can be transfected into a packaging cell line to produce retroviral supernatants and obtain a higher rate of stable cell integration. In addition, this vector contains an incomplete packaging signal and could not produce recombinant retroviruses (The Manual of pSuper RNAi system, OligoEngine, Washington, USA). pSuper.Basic is a pUC origin vector (The Manual of pSuper RNAi system, OligoEngine, Washington, USA) and used to compare the stability with pSuper.Retro in vivo. The difference of both vectors in the gene silencing effects is likely due to their stability in vivo. HBx protein plays an important role in HBV replication and HBV induced HCC [26, 48]. Therefore, HBx became a good target gene for gene silencing in recent years. Importantly, as all HBV RNA transcripts share the same sequence in the HBx due to its unique transcription mechanism, siRNAs targeting the HBx region actually knock down all HBV RNAs, as shown in the previous studies [28, 49, 50].
The result showed that the siRNA targeting the region nt 285–303 of the HBx coding region has a stronger antiviral effect than that targeting the region nt 314–332. It is known that siRNA-specific features such as low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preference, interaction with the cellular machinery called RISC and so on are likely to contribute to efficient RNAi process. The relative influence of these parameters is not fully understood and now under investigation by many groups [51–56]. When pAAV-HBV1.3B and pSB-HBxi285 or pSR-HBxi285 was co-injected into the tail veins of C57BL/6 mice, HBV expression and replication could be significantly inhibited by the first week. Thereafter, the antiviral effect declined in the pSB-HBxi285 treated mice, whereas the antiviral effect of pSR-HBxi285 was maintained for at least 4 weeks. These results indicated that the retroviral vector based HBx siRNA expression plasmid could much more effectively inhibit HBV expression and replication than the non-viral vector based HBx siRNA expression plasmids. Among the mice which received pSR-HBxi285, HBV replication was completely suppressed in the majority of mice, as no HBsAg was detectable for the whole experimental period of 16 weeks. However, HBV gene expression and replication may resume in some mice despite an initial delay, indicating that transient suppression of HBV replication by HBx siRNA may also fail to clear HBV completely in some individuals. It is worth to mention that the change of serum HBsAg and HBV DNA in the mice after the application with HBx siRNAs occurred with different kinetics. The reason may be the different requirement of HBsAg translation and HBV DNA replication. It is well established that only a small fraction of HBsAg is required for virion production . In the patients with chronic HBV infection, HBV DNA loads do not correlated with HBsAg . Our results suggest that measures to prolong the antiviral effect of the siRNA should be investigated in future studies.