Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B

Background It has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI. Methods We constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model. Results The 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective. Conclusions Taken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo.


Introduction
Hepatitis B virus (HBV) causes acute and chronic infection in the human liver and subsequently hepatic cirrhosis and hepatocellular carcinoma (HCC) that severely affects human health [1][2][3]. Although a highly effective vaccine is now available for the prevention of new HBV infections, about 400 million people worldwide have already been chronically infected and suffer from chronic liver injury [4].
An immunologically well-characterized small animal model for HBV infection remains unavailable due to the strict host specificity of HBV infection, which greatly hampers HBV-related research. The laboratory mouse is genetically and immunologically defined, and a large collection of genetically modified animals exists. However, mice can not be infected with HBV. Several lines of transgenic mice with replication competent HBV genomes have been established and represented to be powerful tools for HBV research [5]. However, HBV replication in the transgenic mice is generated from the integrated HBV sequence harbored in all hepatocytes, which is different from what occurs during a natural infection [5][6][7][8]. The presence of HBV genomes in these mouse lines inevitably induces immune tolerance to the HBV antigens. In addition, the capability of production of transgenic mouse model is not readily available in ordinary laboratory conditions. Transplant mouse models were established and used for different studies [9][10][11]. However, the models are based on immunodeficient mouse strains and difficult to handle in the laboratory.
Hydrodynamic injection (HI) of replication-competent HBV DNA into the tail veins of mice can establish HBV replication in the mouse liver [12,13]. In 40% of injected C56BL/6 mice, the persistence of HBV surface antigenemia (HBsAg) was greater than 6 months. HBV persistence is determined by the mouse genetic background and plasmid backbone [12]. The HBV HI mouse model is a highly interesting model for testing vaccination strategies and mechanisms of viral persistence [14][15][16][17][18]. This model may be used to the study replication competence of HBV constructs [15]. Previously, the established HBV HI mouse models were based on HBV genotype A replicationcompetent clones [12,13]. As HBV genotypes B and C are highly prevalent in Asia and may have distinct virological characteristics that correlate with clinical outcomes in the natural course of infection and antiviral therapy, it is warranted to establish HBV HI mouse models with characterized HBV strains.
Patients with chronic HBV infection are currently treated with interferon alpha (IFN-α) or nucleotide analogs such as entecavir and tenofovir. However, the current therapies have only a limited success rate and frequent viral recurrence after cessation of therapy, therefore, new antiviral strategies are required [19,20].
RNA interference has been developed as potential therapeutic approaches [21][22][23][24]. In previous studies, siRNAs targeting different regions of the HBV genome were used. The HBx gene encodes a small non-structural protein that has diverse functions and is required for HBV efficient replication [25,26]. The HBx mRNA contains the common sequence of the four transcripts of HBV [25,27,28], which makes it a potentially useful target for antiviral therapy.
In this study, we addressed the question whether a replication competent HBV genotype B clone could replicate transiently and persistently in HI mouse model. Plasmids pSB-HBxi285 (non-viral vector) and pSR-HBxi285 (retroviral vector) that express siRNA targeting the HBx gene were constructed. Using these constructs, we asked how the gene silencing by HBx siRNA of genotype B influences the course of HBV replication and persistence in the HI mouse model.

Identification of different folds of HBV DNA expression plasmids
The 1.1, 1.2 and 1.3 fold over-length HBV genome DNA including nt 1658-3215-1986, nt 1360-3215-1986 and nt 1040-3215-1986 were cloned into the pBluesript II KS (+) vector separately. The construction procedure is shown in Additional file 1: Figure S1A and schematic representation of the 1.1, 1.2 and 1.3 fold HBV genome are shown in Additional file 1: Figure S1B. The plasmids were analyzed by restriction enzyme digestion with PstI and SacI. PstI and SacI restriction digestion of pBS-HBV1.1 B , pBS-HBV1.2 B and pBS-HBV1.3 B resulted in the appearance of additional bands of 3.5, 3.8 and 4.2 kb in addition to the pBluescript II KS (+) vector, respectively (Additional file 2: Figure S2). Consistently, PstI restriction digestion of pBS-HBV1.1 B, pBS-HBV1.2 B and pBS-HBV1.3 B confirmed that these vectors have the sizes of 6.5, 6.8 and 7.2 kb, respectively (Additional file 2: Figure S2). The HBV 1.3 fold over length genome DNA was amplified and sub-cloned into the NotI site of the pAAV-MCS vector (Agilent technologies, La Jolla, USA). The construction procedure of pAAV-HBV1.3 B is shown in Additional file 1: Figure S1C. The plasmid pAAV-HBV1.3 B was identified by NotI restriction digestion and an additional band of 4.16 kb (data not shown).

pBS-HBV1.3 B shows high replication competence both in vitro and in vivo
To investigate the functionality of pBS-HBV1.1 B , pBS-HBV1.2 B and pBS-HBV1.3 B in hepatoma cells, the plasmids were transfected into Huh-7 cells. At 48 h posttransfection, the culture supernatants of the transfected cells were harvested and subjected to HBsAg and HBeAg detection. As shown in Figure 1A, the HBsAg and HBeAg levels in the culture supernatant of pBS-HBV1.3 B transfected Huh-7 cells were significantly higher than those with the of pBS-HBV1.1 B and pBS-HBV1.2 B plasmids (p < 0.01).
To further compare the replication competence of pBS-HBV1.1 B , pBS-HBV1.2 B and pBS-HBV1.3 B in vivo, BALB/c mice were subjected to HI with these plasmids. Each group contained 10 mice. At 7 dpi, mouse serum samples were collected for HBV DNA quantification. The liver tissue was collected for immunohistochemical staining of HBcAg. Figure 1B showed that HI with pBS-HBV1.3 B resulted in a significantly higher HBV DNA level in mouse serum samples compared with HI with pBS-HBV1.1 B and pBS-HBV1.2 B (p < 0.01). Immunostaining of HBcAg demonstrated that HBcAg positive hepatocytes could be detected in the mouse liver after HI with each of the three plasmids but absent in the control mice. Moreover, the HBcAg positive hepatocytes were most abundant in the mouse liver of pBS-HBV1.3 B transfected mice ( Figure 1C).

The influence of the host genetic background and plasmid backbone on HBV persistence
It is reported that HBV persistence after HI in mice depends on the host genetic background and plasmid backbone [12]. Firstly, the 1.3 fold over-length HBV genome was cloned into pAAV vector to generate pAAV-HBV1.3 B . HI with pAAV-HBV1.3 B was performed in C57BL/6 mice to examine whether this HBV isolate of genotype B is able to establish persistent replication in vivo. In parallel, pAAV-HBV1.2 described by Huang et al. (2006) was used as a reference. pAAV-HBV1.3 B group included 14 mice and pAAV-HBV1.2 group included 9 mice. The serum HBV DNA levels in C57BL/6 mice with pAAV-HBV1.3 B were determined at the indicated time points up to week 48 and were higher than those in mice with pAAV-HBV1.2 ( Figure 2A).
The liver tissue was collected from C57BL/6 mice injected with pAAV-HBV1.3 B at 70, 168 and 252 dpi or pAAV-HBV1.2 at 70 dpi, 168 dpi and 340 dpi. HBV DNA was extracted from the liver tissue samples and assayed for HBV DNA by Southern blot analysis. Bands corresponding to the expected size of the input HBV plasmids and HBV replication intermediates including relaxed circular (RC) and single-stranded (SS) HBV DNAs could be detected in the liver of HBsAg-positive C57BL/6 mice hydrodynamically injected with pAAV-HBV1.3 B or pAAV-HBV1.2 ( Figure 2B). HBV replication intermediates were at a higher level in mice injected with pAAV-HBV1.2 than in those with pAAV-HBV1.3 B. Intrahepatic HBV DNA levels also detected by real-time PCR and the results were consistent with the Southernblot results ( Figure 2C). Immunohistochemical staining of the liver sections for HBcAg revealed that both cytoplasmic and nucleic HBcAg were detected in the liver of HBsAg-positive mice hydrodynamically injected with pAAV-HBV1.3 B or pAAV-HBV1.2 ( Figure 2D). In contrast, no HBcAg positive cell was present in the liver of the control mice and HBsAg-negative mice. All results obtained in this experiment indicate that HBV genotype B construct was replication competent in the mouse liver.
To investigate whether the vector backbone and the host genetic background also influence persistence of the HBV isolate of genotype B, 10 μg of the pBS-HBV1.3 B or pAAV-HBV1.3 B plasmids were injected hydrodynamically into the tail veins of male C57BL/6 or BALB/c mice. Each group included 5 mice. After HI, the mice were regularly bled and the temporal changes in HBsAg, HBeAg, HBcAb, HBsAb and HBV DNA levels were monitored. In BALB/c mice injected with pBS-HBV1.3 B , the HBsAg level increased in the first three days but dropped quickly afterward and all mice became negative at 1 wpi. However, the HBsAg level in BALB/c mice injected with pAAV-HBV1.3 B declined slowly with time and 75% of the mice were HBsAg positive at day 10 dpi. HBsAg became undetectable in all mice at 4 wpi ( Figure 3A). In contrast to BALB/c mice, the HBsAg level decreased much more slowly after injection of the same plasmids in C57BL/6 mice. 50% of C57BL/6 mice injected with pBS-HBV1.3 B were HBsAg-positive at 1 wpi and all mice became negative at 2 wpi. Moreover, 75% of C57BL/6 mice injected with pAAV-HBV1.3 B were still HBsAg-positive at 2 wpi. HBsAg remained detectable in 50% of the mice after 10 weeks ( Figure 3A). These results confirmed that the host genetic background as well as the vector backbone influenced HBV persistence.
In addition, we also compared the kinetics of viremia between pAAV-HBV1.3 B and pAAV-HBV1.2 in the HI C56BL/6 mouse model. The serum HBV DNA level in pAAV-HBV1.3 B injected mice was higher than that in pAAV-HBV1.2 injected mice ( Figure 2A). However, the C57BL/6 mice received pAAV-HBV1.3 B had a lower positive rate of HBsAg compared with that received pAAV-HBV1.2. 20% of the mice are HBsAg positive at week 24 after HI with pAAV-HBV1.3 B , which was only half of that in mice injected with pAAV-HBV1.2 ( Figure 3B).
None of the C57BL/6 mice injected with pAAV-HBV1.2 produced HBsAb after 28 dpi, although all of them produced HBcAb after the day 7 (Table 1). However, 3 of 14 C57BL/6 mice received pAAV-HBV1.3 B produced HBsAb after the 28 dpi. Moreover, the raise of HBsAb in pAAV-HBV1.3 B injected mice was faster than that in pAAV-HBV1.2. As shown in Table 1, for those mice producing HBsAb, they were HBsAg negative. All mice developed HBcAb after 7 dpi. Thus, different replication competent HBV genomes in the AAV vector may differ in their ability to establish the persistent replication in mice.
Impaired HBcAg specific cellular immunity in pAAV-HBV1.2 and pAAV-HBV1.3 B injected C57BL/6 mice during initial activation 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. To confirm this result, HBV-specific CTL responses against full length HBcAg peptide were detected at 3dpi and 10 dpi after HI by ELISPOT assay of IFN-γ producing cells. At 3 dpi we almost could not detect any significant levels of HBcAg specific IFNγ-producing cells in the splenocytes ( Figure 4A). However, the average number of IFNγ secreting cells was 463 and 653 in 1× 10 6 splenocytes in pAAV-HBV1.2 and pAAV-HBV1.3 B injected mice at 10dpi, respectively ( Figure 4B). There is no significant difference between those two groups (p > 0.05). The result indicates that HBcAg-specific immune response may play a key role in clearing HBV infection at early stage in the HI mouse model.

HBxi285 inhibited HBV replication in vitro
To apply RNAi in vivo, the vector based expression of siRNAs was explored in the HI mouse model. For these The antiviral effect of pSR-HBxi314 and pSR-HBxi285 was investigated in vitro. As shown in Figure 5A, the HBsAg and HBeAg levels decreased by 80.2% and 78.7% when pAAV-HBV1.3 B and pSR-HBxi285 were cotransfected into Huh-7 cells, respectively. However, when pAAV-HBV1.3 B and pSR-HBxi314 were co-transfected into Huh-7 cells, the HBsAg and HBeAg levels were only inhibited by 10.5% and 30.3%, respectively. Different concentrations of pSR-HBxi285 were used to test whether the antiviral effect was dose-dependent. Figure 5B and C showed that pSR-HBxi285 inhibited HBsAg and HBeAg expression as well as HBV DNA in a concentrationdependent manner in the range of 0.1-0.5 μg. The 0.5 μg dose of pSR-HBxi285 was most effective for the reduction of HBsAg, HBeAg and HBV DNA in transient transfection.
To compare the antiviral effects of pSB-HBxi285 and pSR-HBxi285 in vitro, pAAV-HBV1.3 B and different concentrations of pSB-HBxi285 or pSR-HBxi285 were cotransfected into Huh-7 cells. Figure 5D shows that pSB-HBxi285 and pSR-HBxi285 inhibited HBsAg and HBeAg in a dose-dependent manner. However, there was no significant difference in their antiviral effect (P > 0.05).
HBxi285 reduced serum HBsAg and HBV DNA levels in C57BL/6 mice after HI Based on the previous experiments, 10 μg of pAAV-HBV1.3 B and 10 μg pSB-HBxi285 or pSR-HBxi285 were co-injected into C57BL/6 mice to determine the antiviral effect of HBxi285 in vivo. Each group contained 10 mice. Initially, HBsAg levels in mice were significantly reduced by HI in the presence of pSB-HBxi285 or pSR-HBxi285 plasmids at 1 dpi (p < 0.05), compared with the control groups receiving empty vectors ( Figure 6A). However, the HBsAg levels in pSB-HBxi285 treated mice quickly increased during the follow up and reached the levels of the control at 14 dpi. In contrast, the HBsAg levels in pSR-HBxi285 treated mice were inhibited to a significantly lower degree before the 28 dpi compared with the control groups (p < 0.05). HBsAg became detectable in some mice at 42 dpi. It is worthy to note that the majority (7 of 10) of the mice received pSR-HBxi285 were HBsAg negative during the whole experimental period ( Table 2). HBV DNA concentrations in mouse sera were determined and were well correlated with HBsAg levels ( Figure 6B). Figure 6C shows that the HBsAg positive rate in pSB-HBxi285 or pSR-HBxi285 treated mice was significantly decreased in comparison to the controls at 1 dpi. Thereafter, the positive rate of HBsAg in pSB-HBxi285 treated mice increased gradually and showed no significant difference in comparison to the control at 7 dpi. The positive rate of HBsAg in pSR-HBxi285 treated mice remained at a lower level (18.2-27.3%) than other groups until the 4th week post injection (wpi) but showed similar levels to the other groups in the 5th wpi. Consistently, immunostaining of HBcAg in the liver section of mice after HI demonstrated that the co-application of pSR-HBxi285 or pBS-HBxi285 abolished HBcAg expression in the mouse liver at 3 dpi ( Figure 6D). However, HBcAg may become detectable in the mouse liver which received pSR-HBxi285 similar to those of control mice 4 weeks after HI. The results indicated that HBxi285 could significantly inhibit the HBsAg production and HBV replication. However the vector pSR-HBxi285 was more efficient in reducing HBsAg and HBV DNA levels than that of pSB-HBxi285.

Discussion
In the present study, an HBV genotype B HI mouse model was established. After HI with pAAV-HBV1.3 B , 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.  (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.  pAAV-HBV1.2 0/9 9/9 0/9 0/9 4/8 The number of C57BL/6 mice in each group positive for anti-HBc or anti-HBs antibodies after injected hydrodynamically with 10 μg of pAAV-HBV1.3 B or pAAV-HBV1.2 plasmids. All the mice with HBsAb are HBsAg negative.
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 [12]. Apparently, HBV replication is not strictly correlated with HBsAg production, as known for patients [34][35][36][37][38]. The result indicated that the model is more robust when pAAV-HBV1.2 genotype A is used as compared with pAAV-HBV1.3 B . 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. Coapplication of the HBx siRNA expression plasmids treatment of these mice induced a significant inhibition of HBV replication and expression. When pAAV-HBV1.3 B 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][42][43][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 [45] 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 Figure 4 Specific T-cell responses to HBcAg epitopes in mice after HI. The specific T-cell responses to the full length HBcAg peptide after HI. The number of HBcAg specific IFNγ secreting cells in 1× 10 6 splenocytes in pAAV-HBV1.2 and pAAV-HBV1.3 B injected mice at 3dpi (A) and 10dpi (B) by IFNγ ELISPOT assay in the presence of 0.5 μg/ml full length HBcAg peptide. The experiments were done in duplicate. The data were analyzed by t test, and there is no significant differences between those two groups (p > 0.05).
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][52][53][54][55][56]. When pAAV-HBV1.3 B 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 [57]. In the patients with chronic HBV infection, HBV DNA loads do not correlated with HBsAg [58]. Our results suggest that measures to prolong the antiviral effect of the siRNA should be investigated in future studies.

Conclusions
In summary, our established HBV genotype B HI mouse model is reliable and could be applied for studies on genetic and antiviral research of chronic HBV infection. The retroviral vector based HBx siRNA expression plasmids could efficiently inhibit HBV expression and replication, suggesting that an siRNA-based treatment strategy may be an additional therapeutic option for future clinical use in patients with chronic HBV infection.

HI experiments
The replication competence of the overlength HBV genomes in vivo was tested by HI with pBS-HBV1.1 B , pBS-HBV1.2 B or pBS-HBV1.3 B plasmids in BALB/c mice (female, 6-8 weeks old, from the breeding colonies of the experimental animal center in Hubei Province, China), with 10 mice per group. C57BL/6 mice (male, 6-8 weeks old, from the breeding colonies of the experimental animal center in Shanghai, China), were subjected to HI with pAAV-HBV1.3 B or pAAV-HBV1.2 to test the ability of these plasmids to establish persistent HBV replication. pAAV-HBV1.3 B group included 14 mice and pAAV-HBV1.2 group included 9 mice. To investigate the factors affecting HBV persistent in the mouse liver, we injected pBS-HBV1.3 B or pAAV-HBV1.3 B into BALB/c or C57BL/6 mice separately, with 5 mice per group. HI experiments were carried out as described previously [17], The quantification of HBV DNA was performed by using a routine real time PCR procedure, described previously [34,[60][61][62][63][64]. The HBV copy numbers were determined by SYBR Green Real time PCR Master Mix (commercially available assay kit, TOYOBO, Osaka, Japan). Melt curve analysis and agarose gel electrophoresis were used to verify the specificity of the real-time PCR. The following primers were used: forward primer: 5′-CTG CAT CCT GCT GCT ATG-3′ (nt 408-425), reverse primer: 5′-CAC TGA ACA AAT GGC AC-3′ (nt 685-701) according to the reference sequence with Genbank accession number (AY220698.1). Serum containing a known concentration of HBV DNA was used as a positive control.

Immunohistochemistry
The liver tissue was taken from the mice receiving HI with pBS-HBV1.1 B , pBS-HBV1.2 B or pBS-HBV1.3 B at 7 day post injection (dpi) and the mice receiving HI with pAAV-HBV1.3 B at 70 dpi, 168 dpi and 252 dpi or pAAV-HBV1.2 at 70 dpi, 168 dpi and 340 dpi, which was then used for immunohistochemical staining of the HBcAg in hepatocytes. The liver tissue of the mice received 0.9% NaCl was used as negative control. Liver tissue was collected from the mice and embedded in paraffin. Intrahepatic HBcAg was visualized by immunohistochemical staining with rabbit anti-HBc (Dako) of the liver tissue sections. The liver tissue sections were also stained with hematoxylin.
Purification and Southern blot analysis of HBV DNA from the liver tissue of mice Total DNA from the pAAV-HBV1.3 B and HBx siRNA expression plasmids co-transfected Huh-7 cells and the liver tissue of mice was isolated using commercial kits (OMEGA, Norcross, USA). The isolated HBV DNA was subjected to agarose gel electrophoresis followed by denaturation and Southern blot analysis. All DNA samples were treated with RNase before gel electrophoresis. HBV DNA was detected by hybridization with a digoxigenin (DIG) (Roche, Lewes, UK)-labeled full length HBV probe. The hybridization signals were visualized on x-ray film using an alkaline phosphatase-anti-DIG-Fab conjugate in the presence of the chemiluminescence substrate CDP-Star (Roche).

Enzyme-Linked Immunospot (ELISPOT) assay
ELISPOT assay was carried out using the mouse IFN-γ precoated ELISPOT Kit (Dakewe, Shenzhen, China) Restriction sites are shown in italics. The gene sequences included in the primers are indicated by underling. a The Genbank accession numbers of the used reference sequences are given.
according to the manufacturer's instructions. Briefly, 96well flat-bottomed microtiter plates were preincubated with the coating antibody (anti-IFN-γ monoclonal antibody) at 4°C overnight and blocked for 2 hr at 37°C. Mouse splenocytes at the density of 1 × 10 6 cells per well were added to wells in triplicate with final concentration of 0.5 μg/ml full length HBcAg peptide (ProSpec, Ness-Ziona, Israel) separately and incubated at 37°C, 5% CO 2 for 24 hr. 1 μg/ml of ConA (Sigma, St. Louis, USA) was used as positive control. Thereafter, cells were removed. Wells were washed ten times with PBS containing 0.05% Tween-20 (PBS-T) and incubated with 100 ml of biotinylated anti-IFNγ antibody for 1 hr. The plates were washed again with PBS-T and incubated with 50 ml HRP-streptoavidin solution at 37°C for 1 hr. Spot-forming cells were counted and analyzed with an ELISPOT plate reader (BioReader 4000, Biosys, Germany). Results were presented as spot-forming cells per 1 × 10 6 cells. The above complementary sequences were phosphorylated and ligated into BglII and HindIII digested pSuper.Basic or pSuper.Retro vector. The resulting plasmids were named as pSB-HBxi314, pSB-HBxi285, pSB-HBxicon, pSR-HBxi314, pSR-HBxi285 and pSR-HBxicon. The clones were sequenced to confirm the correctness of the sequences.

Statistical analysis
The data were evaluated by Student's t-test or one-way ANOVA followed by Tukey's post hoc test. The statistical significance of the data was considered at p < 0.05.