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BACKGROUND & AIMS Early determination of the prognosis of patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is important to guide clinical management and decrease mortality. The aim of this study was to develop a new simplified prognostic score to accurately predict outcomes in patients with HBV-ACLF. METHODS Prospective clinical data from 2,409 hospitalized patients with acute deterioration of HBV-related chronic liver disease were used to develop a new prognostic score that was validated in an external group. RESULTS A total of 954 enrolled patients with HBV-ACLF were diagnosed based on the Chinese Group on the Study of Severe Hepatitis B-ACLF (COSSH-ACLF) criteria. Six predictive factors were significantly related to 28-day mortality and constituted a new prognostic score (=1.649×ln(international normalized ratio)+0.457×hepatic encephalopathy score+0.425×ln(neutrophil)+0.396×ln(total bilirubin)+0.576×ln(serum urea)+0.033×age). The C-indices of the new score for 28-/90-day mortality (0.826/0.809) were significantly higher than those of 4 other scores (COSSH-ACLF, 0.793/0.784; CLIF-C ACLF, 0.792/0.770; MELD, 0.731/0.727; MELD-Na, 0.730/0.726; all p

The susceptibility of mice to hepatitis B virus (HBV) infection depends on their genetic background. The gut microbiota modulates the antiviral immune response in the liver and plays a protective role against HBV infection. However, whether HBV infection outcomes depend on the gut microbiota remains unclear. In this study, we assessed the gut microbiota composition in naïve BALB/c and C57BL/6 mice using 16S rRNA gene sequencing. The gut microbiota in BALB/c mice was depleted using broad-spectrum antibiotics (ABX) and then reconstituted with fecal microbiota from naïve BALB/c or C57BL/6 mice to evaluate the effect of fecal microbiota transplantation (FMT) on the outcomes of and immune response to HBV infection. We found that HBV infection outcomes and the gut microbiota composition differed between BALB/c and C57BL/6 mice. Commensal bacteria from the fecal microbiota selectively colonized the guts of ABX-treated BALB/c mice. Mice receiving fecal microbiota from BALB/c or C57BL/6 mice displayed different HBV infection outcomes. The fecal microbiota from C57BL/6 mice induced immune tolerance in the liver and prolonged HBV infection. In conclusion, HBV infection outcomes in mice are determined by the host genetic background and gut microbiota composition. Reconstitution of the gut microbiota by FMT can alter the susceptibility to HBV infection in mice.

Commensal gut microbiota protects the immune defense of extra-intestinal organs. Gut microbiota depletion by antibiotics can impair host antiviral immune responses and alter hepatitis B virus (HBV) infection outcomes. However, how gut microbiota modulates antiviral immune response in the liver remains unclear. Here, mice were treated with broad-spectrum antibiotics to deplete gut microbiota. Gut integrity was evaluated, and translocation of live commensal gut bacteria and their components into the liver was investigated. An HBV infection model was established to evaluate impairment of antiviral immune response in the liver after gut microbiota depletion. We found that gut microbiota depletion was associated with impairment of colon epithelial integrity, and live commensal gut microbiota could translocate to the liver. Further, T cell antiviral function in the liver was impaired, partially relying on enhanced PD-1 expression, and HBV immune clearance was hampered. In conclusion, gut microbiota depletion by antibiotics can impair gut barrier function and suppress T cell antiviral immune response in the liver.

Functional maturation of liver sinusoidal endothelial cells (LSECs) plays an important role in intrahepatic T‐cell activation and control of viral infections. Natural killer (NK) cells have been reported to prompt the maturation of antigen‐presenting cells (APCs), especially for dendritic cells (DCs), but the interaction between NK cells and LSECs is elusive. Here, we investigated whether and how NK cells are involved in regulating LSEC maturation and if this has a role in controlling hepatitis B virus (HBV) infection in a mouse model. A chronic HBV replication mouse model was established by hydrodynamic injection (HI) of 6 µg adeno‐associated virus plasmid (pAAV)/HBV 1.2. The nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) ligand diaminopemelic acid (DAP) was imported into liver by HI at day 14 after plasmid injection. We found that HI of DAP recruited conventional NK cells (cNK) into the liver and promoted tumor necrosis factor alpha (TNF‐α) and interferon‐gamma (IFN‐γ) production of NK cells in a chemokine (C‐X‐C motif) receptor 3 (CXCR3)‐dependent manner. Importantly, the maturation of LSECs and the anti‐HBV effects of DAP were impaired in CXCR3−/− mice; this possibly was associated with the decreased number of intrahepatic cNK cells. Consistently, depleting cNK cells but not liver‐resident NK cells also impaired the maturation and antigen‐presenting function of LSECs, which reduced intrahepatic HBV‐specific T‐cell responses and thus inhibited HBV clearance both in wild‐type and in Rag1−/− mice. Moreover, TNF‐α or IFN‐γ stimulation as well as coculture with intrahepatic NK cells partly promoted LSEC phenotypic and functional maturation in vitro. Conclusion: NOD1‐triggered NK cell activation may lead to the enhancement of intrahepatic T‐cell responses by promoting maturation of LSECs through soluble cytokines and cell–cell contact, thereby controlling HBV replication and expression., NK cells promote intrahepatic T cell responses by regulating LSEC maturation. The potential mechanisms by which NK cells regulate the maturation of LSECs may involve soluble cytokines as well as cell‐cell contact. The recruitment and activation of conventional NK cells (cNK) cells after NOD1 activation depend on CXCR3 axis.

It remains controversial how interferon (IFN) response contributes to hepatitis B virus (HBV) control and pathogenesis. A previous study identified that hydrodynamic injection (HI) of type I IFN (IFN-I) inducer polyinosinic-polycytidylic acid (poly(I:C)) leads to HBV clearance in a chronic HBV mouse model. However, recent studies have suggested that premature IFN-I activation in the liver may facilitate HBV persistence. In the present study, we investigated how the early IFN-I response induces an immunosuppressive signaling cascade and thus causes HBV persistence. We performed HI of the plasmid adeno-associated virus (pAAV)/HBV 1.2 into adult BALB/c mice to establish an adult acute HBV replication model. Activation of the IFN-I signaling pathway following poly(I:C) stimulation or murine cytomegalovirus (MCMV) infection resulted in subsequent HBV persistence. HI of poly(I:C) with the pAAV/HBV 1.2 plasmid resulted in not only the production of IFN-I and the anti-inflammatory cytokine interleukin (IL)-10 but also the expansion of intrahepatic regulatory T cells (Tregs), Kupffer cells (KCs) and myeloid-derived suppressor cells (MDSCs), all of which impaired the T cell response. However, when poly(I:C) was injected at day 14 after the HBV plasmid injection, it significantly enhanced HBV specific T cell responses. In addition, interferon-alpha/beta receptor (IFNAR) blockade rescued T cell response by downregulating of IL-10 expression and decreasing Treg and KC expansion. Consistently, Treg depletion or IL-10 blockade also controlled HBV replication. Importance: IFN-I plays a double-edged sword role during chronic HBV infection. Here, we identified that application of IFN-I at different time points causes contrast outcome. Activation of the IFN-I pathway before HBV replication induces an immunosuppressive signaling cascade in the liver, and consequently caused HBV persistence while IFN-I activation post HBV infection enhances HBV-specific T cell responses and thus promote HBV clearance. This result provided an important clue to the mechanism of HBV persistence in adult individuals.

Th17 responses have been shown to play crucial roles in the pathogenesis of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF). The mechanism underlying the enhanced Th17 responses in these patients remains largely unclear. Here we investigated toll-like receptors (TLRs) expression in peripheral T cells and their roles in Th17 cell differentiation and disease aggravation in ACLF patients. 18 healthy subjects (HS), 20 chronic HBV-infected (CHB) patients, and 26 ACLF patients were enrolled and examined for TLRs expression in peripheral blood mononuclear cells (PBMCs). The correlations of T cell TLR2 expression with the antigen non-specific Th17 responses and disease aggravation, as well as the Th17 response to TLR2 ligand stimulation were evaluated in ACLF patients. Compared to HS and CHB patients, ACLF patients showed a distinct TLRs expression pattern in PBMCs. Significantly increased TLR2 expression in T cells was observed in ACLF patients. The TLR2 expression in CD4+ T cells was correlated with the Th17 responses and the clinical markers for disease aggravation in ACLF patients. Moreover, TLR2 ligands stimulation promoted Th17 cell differentiation and response in PBMCs of ACLF patients. These findings implicate that TLR2 signaling plays critical roles in Th17 cell differentiation and disease aggravation of HBV-related ACLF. OA gold

Gut microbiota composition is known to be associated with the progression of hepatitis B virus (HBV)-related liver cirrhosis in humans, outcome of HBV infection in mice, and seroconversion of HBV e-antigen in nucleot(s)ide analog-treated patients. The dynamic alteration of the gut microbiota following HBV infection is still unknown. In this study, a hydrodynamic injection mouse model mimicking acute or chronic HBV infection in humans with comparable virological and immunological features was used. The composition of gut microbiota in the control mice and mice with acute or chronic HBV infection was analyzed at different time points using the Illumina MiSeq platform. The expression of immune molecules in the colon was detected by real-time polymerase chain reaction. We found that the changes in gut microbiota composition, including the total operational taxonomic unit (OTU) count and Shannon-Weaver index, were significantly delayed in mice with HBV infection. Furthermore, the ratio of Bacteroidetes and Firmicutes was stable in the control mice, whereas remarkable dynamic patterns were observed in mice with HBV infection. Interestingly, the dynamic changes in Lactobacillus and Bifidobacterium were found to differ in acute or chronic HBV infection. In addition, the expression of IFN-γ and PD-L1 in the colon was found to be up-regulated early in mice with acute HBV infection, whereas the expression of PD-L1 in the colon of mice with chronic HBV infection was up-regulated later. These data indicate that HBV infection could hamper the development of the gut microbiota community and dynamically change the gut Firmicutes/Bacteroidetes ratio. These data improve our understanding of the relationship between gut microbiota and HBV infection.

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

Background & Aims Multidrug resistance protein 4 (MRP4) has been associated with nucleos(t)ide analogue (NA) antiretroviral therapy failure, though is unclear if MRP4 is also correlated with the failure of anti-hepatitis B virus (HBV) therapy. Methods Multidrug resistance protein 4 expression in human peripheral blood mononuclear cells (PBMCs), liver tissues and human hepatoma cell lines was detected by real-time polymerase chain reaction (PCR), western blotting and immunohistochemistry assays. Supernatant and intracellular HBV DNA levels of MRP4-overexpressing or silenced HepG2.4D14 (wild-type) and HepG2.A64 (entecavir-resistant mutant) cells were measured by quantitative PCR. NA concentrations and HBV mutational analysis were assessed by liquid chromatography/mass spectrometry assays and DNA sequencing. Multivariate analysis was used to assess predictive factors for treatment failure. Results High expression of MRP4 was found in hepatoma cell lines and PBMCs, and up- or down-regulation of MRP4 expression altered the susceptibility of cells to NAs. MRP inhibitors increased NA intracellular accumulation and decreased extracellular levels. Moreover, MRP4 expression in PBMCs was correlated with that in paired liver tissues. Furthermore, multivariate analysis showed MRP4 mRNA expression to be an independent predictor of NA treatment failure. Conclusions Multidrug resistance protein 4 is a critical protein associated with the antiviral efficacy of NAs, and combination therapy of NA and MRP inhibitors could reduce the dosage for long-term NA use. This is the first report to demonstrate that MRP4 expression is an important factor predicting treatment failure in chronic hepatitis B patients and will provide a potential therapeutic target against HBV.

Nucleic acid polymers (NAPs) block the release of HBsAg from infected hepatocytes. These compounds have been previously shown to have the unique ability to eliminate serum surface antigen in DHBV-infected Pekin ducks and achieve multilog reduction of HBsAg or HBsAg loss in patients with chronic HBV infection and HBV/HDV coinfection. In ducks and humans, the blockage of HBsAg release by NAPs occurs by the selective targeting of the assembly and/or secretion of subviral particles (SVPs). The clinically active NAP species REP 2055 and REP 2139 were investigated in other relevant animal models of HBV infection including woodchucks chronically infected with WHV, HBV transgenic mice and HBV infected SCID-Hu mice. The liver accumulation of REP 2139 in woodchucks following subcutaneous administration was examined and was found to be similar to that observed in mice and ducks. However, in woodchucks, NAP treatment was associated with only mild (36–79% relative to baseline) reductions in WHsAg (4/10 animals) after 3–5 weeks of treatment without changes in serum WHV DNA. In HBV infected SCID-Hu mice, REP 2055 treatment was not associated with any reduction of HBsAg, HBeAg or HBV DNA in the serum after 28 days of treatment. In HBV transgenic mice, no reductions in serum HBsAg were observed with REP 2139 with up to 12 weeks of treatment. In conclusion, the antiviral effects of NAPs in DHBV infected ducks and patients with chronic HBV infection were weak or absent in woodchuck and mouse models despite similar liver accumulation of NAPs in all these species, suggesting that the mechanisms of SVP assembly and or secretion present in rodent models differs from that in DHBV and chronic HBV infections.

Liver sinusoidal endothelial cells (LSECs) are organ resident APCs capable of antigen presentation and subsequent tolerization of T cells under physiological conditions. In this study, we investigated whether LSEC pretreatment with NOD-like receptor (NLR) agonists can switch the cells from a tolerogenic to an immunogenic state and promote the development of T cell immunity. LSECs constitutively express NOD1, NOD2 and RIPK2. Stimulation of LSECs with DAP induced the activation of NF-κB and MAP kinases and upregulated the expression of chemokines (CXCL2/9, CCL2/7/8) and cytokines (IFN-γ, TNF-α and IL-2). Pretreatment of LSECs with DAP induced significantly increased IFN-γ and IL-2-production by HBV-stimulated CD8+ T cells primed by DAP-treated LSECs. Consistently, a significant reduction in the HBV DNA and HBsAg level occurred in mice receiving T cells primed by DAP-treated LSECs. MDP stimulation had no impact on LSECs or HBV-stimulated CD8+ T cells primed with MDP-treated LSECs except for the upregulation of PD-L1. DAP stimulation in vitro could promote LSEC maturation and activate HBV-specific T cell responses. These results are of particular relevance for the regulation of the local innate immune response against HBV infections.

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.

We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro . Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo . The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. IMPORTANCE It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2 and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-β were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1β were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-β stimulated by VACV ds 70 mer and AIM2 siRNA interference downregulated AIM2 and IL-1β transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-β and IL-1β, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.

Transforming growth factor beta (TGF-β) is an important cytokine with pleiotropic regulatory functions in the immune system and in the responses against viral infections. TGF-β acts on a variety of immune cells through the cell surface TGF-β receptor (University of Duisburg-EssenTGFBR). The woodchuck has been used as a biomedical model for studies of obesity and energy balance, endocrine and metabolic function, cardiovascular, cerebrovascular and neoplastic disease. Woodchucks infected with woodchuck hepatitis virus (WHV) represent an informative animal model to study hepatitis B virus (HBV) infection. In this study, the cDNA sequences of woodchuck TGF-β1, TGF-β2, TGFBR1 and TGFBR2 were cloned, sequenced and characterized. The full-length TGFBR1 cDNA sequence consisted of 1305bp coding sequence (CDS) that encoded 434 amino acids with a molecular weight of 48.9kDa. The phylogenetic tree analysis revealed that the woodchuck TGF-β family genes had a closer genetic relationship with Ictidomys tridecemlineatus. One antibody with cross-reactivity to woodchuck TGFBR1 was identified by flow cytometry. Moreover, the expression of these genes were analyzed at the transcriptional level. The quantitative PCR analysis showed that the TGF-β family transcripts were constitutively expressed in many tissues tested. Altered expression levels of the TGF-β family transcripts in the liver of WHV infected woodchucks were observed. These results serve as a foundation for further insight into the role of the TGF-β family in viral hepatitis in woodchuck model. Our work also possesses the potential value for characterizing the TGF-β family in other related diseases, such as obesity-related diseases, metabolic disorder, cardiovascular disease and cancer.

Regulatory T cells (Tregs) play an important role in counter-regulating effector T cell responses in many infectious diseases. However, they can also contribute to the development of T cell dysfunction and pathogen persistence in chronic infections. Tregs have been reported to suppress virus-specific T cell responses in hepatitis B virus (HBV) infection of human patients as well as in HBV animal models. However, the phenotype and expansion of Tregs has so far only been investigated in other infections, but not in HBV. We therefore performed hydrodynamic injections of HBV plasmids into mice and analyzed the Treg response in the spleen and liver. Absolute Treg numbers significantly increased in the liver but not the spleen after HBV injection. The cells were natural Tregs that surprisingly did not show any activation or proliferation in response to the infection. However, they were able to suppress effector T cell responses, as selective depletion of Tregs significantly increased HBV-specific CD8+ T cell responses and accelerated viral antigen clearance. The data implies that natural Tregs infiltrate the liver in HBV infection without further activation or expansion but are still able to interfere with T cell mediated viral clearance. CA extern

Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

The interferon-alpha (IFN-alpha) gene family is an important part of the immune system. Recombinant interferon-alpha is widely used to treat viral hepatitis and malignant diseases. Marmota himalayana has been found to be susceptible to woodchuck hepatitis virus, a virus genetically related to hepatitis B virus (HBV), and is suitable as an animal model for studies on HBV infection. Here, the IFN-alpha gene family of M. himalayana (cwIFN-alpha) was characterized. Sequence data indicate that the cwIFN-alpha family consists of at least 8 functional sequences and 6 pseudogenes with high homology within the family and to IFN-alpha of Marmota monax, a related species and well-established animal model. The recombinant cwIFN-alpha subtypes were expressed and tested to be active in viral protection assay and to induce expression of MxA in a species-specific manner. This work provides essential information for future work on testing new therapeutic approaches of HBV infection based on IFN-alpha in M. himalayana.

To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg, HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4(+) and CD8(+) T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4(+) and CD8(+) T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4(+) T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

T helper type 1 (Th1) immunity was considered to play a dominant role in viral clearance of hepadnaviral infection. However, pre-primed Th2 type responses were able to efficiently control hepadnaviral infection in animal models. We investigated how pre-primed Th1/2 responses control hepadnaviral replication using the newly established mouse models. DNA (pWHcIm, pCTLA-4-C) and protein vaccines based on the nucleocapsid protein (WHcAg) of woodchuck hepatitis virus (WHV) primed specific immune responses with distinct features. The pre-primed responses determined the characteristics of recall responses if challenged with a WHcAg-expressing adenoviral vector. Vaccination with pWHcIm and pCTLA4-C facilitated viral control in the hydrodynamic injection model and reduced WHV loads by about 3 and 2 logs in WHV-transgenic mice, respectively, despite of different kinetics of specific CD8+ T cell responses. Thus, pre-primed Th2-biased responses facilitate the development of CD8+ T cell responses in mice compared with naïve controls and thereby confer better viral control. OA embargo

Objectives The current strategies for hepatitis B virus (HBV) post-exposure prophylaxis (PEP) are not generally available in remote and rural areas of developing countries and/or carry potential risks for infection with blood-borne transmitted pathogens. Nucleotide analogues (NAs) are successfully used for human immunodeficiency virus PEP, and maybe effective for HBV PEP. In this study, we tested the NA-based strategies for HBV PEP using the Chinese woodchuck model. Methods Chinese woodchucks were inoculated intravenously with different doses of woodchuck hepatitis virus (WHV). A deoxyguanosine analogue entacavir (ETV), a DNA vaccine pWHcIm, or ETV plus pWHcIm were applied to the infected animals 24 h later. Twenty weeks later, the animals were re-challenged with WHV to test for the presence of immunity against WHV. Results Inoculation with different WHV doses had a strong influence on the course of WHV infection; NA alone or in combination with a DNA vaccine completely prevented viremia after a high dose of WHV inoculation in Chinese woodchucks and induced partial or complete protective immunity, respectively. Conclusions NA-based PEP strategies (NA alone or in combination with vaccine) may be an alternative of HBV PEP, especially in those living in the remote and rural areas of the developing countries and the non-responders to the current vaccine, and may be valuable in the PEP of HBV and HIV co-infection after occupational and non-occupational exposure. Further clinical studies are warranted to confirm the valuable of NA-based strategies in HBV PEP.

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamidetreated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression. © 2014 Wang et al. OA gold

Interferon alpha (IFN-α) is commonly used for the treatment of chronic hepatitis B (CHB) patients. Many factors including viral genetics may determine the outcome of IFN-α therapy. In this study, we tested whether the expression of IFN-α directly in the liver inhibits HBV gene expression and replication using a HBV hydrodynamic injection (HI) mouse model. Two replication-competent clones from different HBV isolates that belonging to HBV genotype A and B based on a pAAV vector (pAAV-HBV-A and pAAV-HBV-B) were compared for their susceptibility to IFN-α. HBV clones were injected into mice either alone or in combination with a murine (m) IFN-α expression plasmid (pmIFN-α). HBsAg and HBeAg concentrations and HBV DNA levels in mice differed after injection of these two HBV clones. Co-application of pmIFN-α together with the two distinct isolates resulted in markedly different kinetics of decline of HBsAg, HBeAg, and HBV DNA levels in the mice. Immunohistochemical staining of liver sections with anti-HBc showed that mIFN-α application completely inhibited the expression of HBcAg in mice inoculated with pAAV-HBV-B, whereas the expression of HBcAg was only reduced in mice with pAAV-HBV-A. Consistently, mice injected with pAAV-HBV-B and pmIFN-α showed higher expression levels of the IFN-stimulated genes (ISGs) ISG15, OAS, PKR as well as proinflammatory cytokine IL-6 in the liver. In addition, expression levels of anti-inflammatory cytokine IL-10 was down-regulated significantly in liver of the mice injected with pAAV-HBV-B and pmIFN-α. Our data demonstrate that IFN-α exerts antiviral activity in HBV mouse model, but different HBV isolates may have diverse susceptibility to IFN-α.

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.

Interleukin 10 (IL-10) is a pleiotropic cytokine acting on a variety of immune cells through the cell surface receptor (IL-10R). It has been suggested to resuscitate antiviral immunity by interfering with IL-10/IL-10R pathway. The woodchuck model infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study hepatitis B virus (HBV) infection. In this study, the woodchuck IL-10R (wIL-10R) was molecularly cloned and characterized, showing high similarity of its nucleotide and amino acid sequences to that of other mammalian species. The expression level of wIL-10R mRNA in woodchuck peripheral blood mononuclear cells was significantly increased in acute WHV infection but down-regulated during chronic WHV infection. Specific rabbit antibodies against wIL-10R were prepared and showed the ability to enhance the proliferation and degranulation of specific T-cells from chronically WHV-infected woodchucks in vitro. The present work on wIL-10R provided a good basis for future preclinical studies on therapeutic approaches for chronic HBV infection.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an inhibitory T cell receptor predominately expressed on activated T cells and plays an important role in regulation of specific T cell responses to viral infection. The woodchuck model is an informative animal model for hepatitis B virus (HBV) infection. In this study, the extracellular region of woodchuck CTLA-4 (wCTLA-4) was cloned and the fusion protein of GST-wCTLA-4 was expressed and purified. Polyclonal antibody against GST-wCTLA-4 (anti-GST-wCTLA-4) was prepared. The full length wCTLA-4 protein expressed in transfected baby hamster kidney cells was detected by anti-GST-wCTLA-4 in western blot analysis and immunofluorescence staining. Anti-GST-wCTLA-4 provides a useful tool to study the role of CTLA-4 in T-cell response in the woodchuck model. Further, the blocking of CTLA-4 with anti-GST-wCTLA-4, as a novel therapy approach for chronic hepatitis B virus infection, could be studied in woodchuck model now.

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.

AIM: To investigate the expression of programmed death (PD)-1, PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC). METHODS: Liver biopsies and HCC specimens from patients were collected and histologically examined. The expression of PD-1, PD-L1, and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining. The association between the expression level of PD-1, PD-L1, and PD-L2 and clinical and pathological variables was analyzed statistically. RESULTS: Expression of PD-1 was found in liver-infiltrating lymphocytes. In contrast, PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells. The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756, P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001, P = 0.018). PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051, P = 0.000; 1.05 ± 1.099 vs 4.29 ± 3.885, P = 0.004; 1.80 ± 1.473 vs 3.81 ± 3.400, P = 0.020). CONCLUSION: PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B. PD-1 and PD-Ls may play a role in immune evasion of tumors.

Background: The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV). Methods: Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined. Principal Findings: Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks. Conclusions: Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection. © 2011 Zhang et al.

Hepatitis B virus infection (HBV) is a major medical problem in China. The lack of a suitable infection model in China is recognized as an obstacle for research on HBV in China. Chinese Marmota-species is phylogenetically closely related to Marmota monax, thus, it might be suitable to serve as an animal model for HBV infection. Therefore, we attempted to prove the claim about the existence of woodchuck hepatitis virus (WHV)-like viruses in Chinese Marmota-species and to determine the susceptibility of these species to experimental WHV infection. In the present study, 653 sera from three Chinese Marmota-species, Marmota himalayana, Marmota baibacina and Marmota bobak, were screened for WHV-like viruses by serological and molecular assays. The susceptibility to WHV of three species was investigated by experimental infection and monitored by testing of anti-WHc and WHsAg by ELISA, detection of WHV DNA by PCR, and detection of WHV replication intermediates and antigens in liver samples. No evidence for the existence of a genetically closely related virus to WHV in three Chinese Marmota-species was found by serological assays and PCR. M. himalayana was susceptible to WHV infection as inoculated animals became positive for anti-WHc, WHsAg and WHV DNA. Further, WHV replication intermediates and proteins were detected in liver samples. In contrast, M. baibacina remained negative for tested virological parameters. M. bobak species showed a limited susceptibility to WHV. Our data do not support early reports about WHV-like viruses in China. M. himalayana is suitable for the establishment of a model for hepadnaviral infection.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Hepatitis B virus (HBV)-associated nephritis has been reported worldwide. Immune complex deposition has been accepted as its pathogenesis, although the association between the presence of local HBV DNA and viral antigen and the development of nephritis remains controversial. To understand better the roles played by HBV protein expression in the kidney, the global gene expression profile was studied in the kidney tissue of a lineage of HBV transgenic mouse (#59). The mice expressed HBsAg in serum, and HBsAg and HBcAg in liver and kidney, but without virus replication. Full-length HBV genome (adr subtype, C genotype) isolated from a chronic HBV carrier was used to establish the transgenic mice #59. Similarly manipulated mice that did not express HBV viral antigens served as controls. Southern blotting, hybridization with HBV probe, and immuno-histochemical staining were used to study HBV gene expression. mRNA extracted from the kidney tissue was analyzed using Affymetrix microarrays. HBsAg and HBcAg were located mainly in the cytoplasm of tubular epithelium. Altogether 520 genes were "up-regulated" more than twofold and 76 genes "down-regulated" more than twofold in the kidney. The complement activation, blood coagulation, and acute-phase response genes were markedly "up-regulated". Compared to the controls, the level of serum C3 protein was decreased in #59 mice, while the level of C3 protein from kidney extract was increased. Results indicate that expression of HBsAg and HBcAg in tubular epithelial cells of the kidney per se can up-regulate complement-mediated inflammatory gene pathways, in addition to immune complex formation.

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8 + memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PDL1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients. Citation: Liu J, Zhang E, Ma Z, Wu W, Kosinska A, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856 Editor: Charles M. Rice, The Rockefeller University, United States of America Received April 17, 2013; Accepted November 13, 2013; Published January 2, 2014 Copyright: 2014 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (TRR60 and GK 1045/2), National Major Science and Technology Project for Infectious Diseases of China (2008ZX10002-011, 2012ZX10004503), the National Natural Science Foundation of China (No. 30271170, 30571646, 81101248), and the International Science & Technology Cooperation Program of China (2011DFA31030). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michael.roggendorf@uni-due.de

AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients. METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap I digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel. RESULTS: A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent. CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.