Search

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

The Eastern woodchuck (Marmota monax) is a classical animal model for studying hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) in humans. Recently, we found that Marmota himalayana, an Asian animal species closely related to Marmota monax, is susceptible to woodchuck hepatitis virus (WHV) infection and can be used as a new mammalian model for HBV infection. However, the lack of genomic sequence information of both Marmota models strongly limited their application breadth and depth. To address this major obstacle of the Marmota models, we utilized Illumina RNA-Seq technology to sequence the cDNA libraries of liver and spleen samples of two Marmota monax and four Marmota himalayana. In total, over 13 billion nucleotide bases were sequenced and approximately 1.5 billion clean reads were obtained. Following assembly, 106,496 consensus sequences of Marmota monax and 78,483 consensus sequences of Marmota himalayana were detected. For functional annotation, in total 73,603 Unigenes of Marmota monax and 78,483 Unigenes of Marmota himalayana were identified using different databases (NR, NT, Swiss-Prot, KEGG, COG, GO). The Unigenes were aligned by blastx to protein databases to decide the coding DNA sequences (CDS) and in total 41,247 CDS of Marmota monax and 34,033 CDS of Marmota himalayana were predicted. The single nucleotide polymorphisms (SNPs) and the simple sequence repeats (SSRs) were also analyzed for all Unigenes obtained. Moreover, a large-scale transcriptome comparison was performed and revealed a high similarity in transcriptome sequences between the two marmota species. Our study provides an extensive amount of novel sequence information for Marmota monax and Marmota himalayana. This information may serve as a valuable genomics resource for further molecular, developmental and comparative evolutionary studies, as well as for the identification and characterization of functional genes that are involved in WHV infection and HCC development in the woodchuck model. CA extern

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T2A-1 and T1c-19 expressing Cry2A* and Cry1C* from ‘Minhui 63’ (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T2A-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT)50 of larvae fed with T2A-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T2A-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T2A-1 Bt rice lines.

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 effects of fenofibrate on the kidneys of diabetic rats were investigated by measuring the inflammatory responses associated with transcription factor nuclear factor kappa-B (NF-kappa B) pathway.Male Wistar rats were randomly divided into 3 groups: normal control, diabetes control and diabetes + fenofibrate (10 in each group). The expression of NF-kappaB p65, plasminogen activator inhibitor-1 (PAI-1), and intercellular adhesion molecule-1 (ICAM-1) in renal cortex was detected by Western blot, RT-PCR, and immunohistochemistry, respectively. Blood lipid profiles, glucose, and urine albumin were measured as well.The expression of NF-kappa B p65, PAI-1, and ICAM-1 was significantly higher in the diabetes control than those in the normal control, and treatment with fenofibrate inhibited the increased expression of these factors in kidneys by 48.18 %, 35.04 %, and 26.41 %, respectively when compared with the diabetes control, although they were still higher in diabetes + fenofibrate than those in the normal control. Correspondingly, the profiles of lipid were significantly elevated in the diabetes control compared with the normal control, and decreased significantly in diabetes + fenofibrate.Fenofibrate exhibited a downregulating effect on the NF-kappa B pathway in diabetic kidneys, implying that fenofibrate could be a potential treatment for diabetic nephropathy.

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.

The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

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.

Simulated emission depletion (STED) microscopy is very powerful, but still suffers from small tissue penetration depth, photobleaching of fluorescent probes and complicated imaging systems. Here, we propose an optical luminescence depletion mechanism employing upconverting nanoparticles (UCNPs) and explore its potential for multi-photon STED-like microscopy. With the addition of Yb3+ ions in NaYF4:Er3+ UCNPs, the two-photon green emission of Er3+ under 795-nm excitation was successfully depleted by 1140-nm laser through the synergetic effect of the excited state absorption and the interionic energy transfer. This STED-like depletion mechanism was systematically investigated using steady-state rate equations, evidenced by the surprising emerging of 478-nm emission. The green emission depletion efficiency was about 30%, limited by the current laser source. Our work indicates that NaYF4:Yb3+/Er3+ UCNPs will be potential probes for multi-photon super-resolution microscopy with many advantages, including long-wavelength-induced large penetration, non-photobleaching and non-photoblinking properties, cost-effective and simplified imaging systems.

Grapevine is one of the most economically important and widely cultivated fruit crop worldwide. With the industrialization and the popular application of cupric fungicides in grape industry, copper stress and copper pollution are also the factors affecting grape production and berry and wine quality. Here, 3,843 transcripts were significantly differently expressed genes in response to Cu stress by RNA-seq, which included 1,892 up-regulated and 1,951 down-regulated transcripts. During this study we found many known and novel Cu-induced and -repressed genes. Biological analysis of grape samples were indicated that exogenous Cu can influence chlorophylls metabolism and photosynthetic activities of grapevine. Most ROS detoxification systems, including antioxidant enzyme, stress-related proteins and secondary metabolites were strongly induced. Concomitantly, abscisic acid functioned as a negative regulator in Cu stress, in opposite action to ethylene, auxin, jasmonic acid and brassinolide. This study also identified a set of Cu stress specifically activated genes coding copper transporter, P1B-type ATPase, multidrug transporters. Overall, this work was carried out to gain insights into the copper-regulated and stress-responsive mechanisms in grapevine at transcriptome level. This research can also provide some genetic information that can help us in better vinery management and breeding Cu-resistant grape cultivars.

Fruit ripening is a complex process that is regulated by a signal network. Whereas the regulatory mechanism of abscisic acid has been studied extensively in non-climacteric fruit, little is know about other signaling pathways involved in this process. In this study, we performed that plant hormone jasmonic acid plays an important role in grape fruit coloring and softening by increasing the transcription levels of several ripening-related genes, such as the color-related genes PAL1, DFR, CHI, F3H, GST, CHS, and UFGT; softening-related genes PG, PL, PE, Cell, EG1, and XTH1; and aroma-related genes Ecar, QR, and EGS. Lastly, the fruit anthocyanin, phenol, aroma, and cell wall materials were changed. Jasmonic acid positively regulated its biosynthesis pathway genes LOS, AOS, and 12-oxophytodienoate reductase (OPR) and signal pathway genes COI1 and JMT. RNA interference of grape jasmonic acid pathway gene VvAOS in strawberry fruit appeared fruit un-coloring phenotypes; exogenous jasmonic acid rescued this phenotypes. On the contrary, overexpression of grape jasmonic acid receptor VvCOI1 in the strawberry fruit accelerated the fruit-ripening process and induced some plant defense-related gene expression level. Furthermore, jasmonic acid treatment or strong jasmonic acid signal pathway in strawberry fruit make the fruit resistance against Botrytis cinerea.

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.

The microRNA171 (miR171) family is widely distributed and highly conserved in a range of species and plays critical roles in regulating plant growth and development through repressing expression of SCARECROW-LIKE (SCL) transcription factors. However, information on the evolutionary conservation and functional diversification of the miRNA171 family members remains scanty. We reconstructed the phylogenetic relationships among miR171 precursor and mature sequences so as to investigate the extent and degree of evolutionary conservation of miR171 in Arabidopsis thaliana (L.) Heynh. (ath), grape (Vitis vinifera L.) (vvi), poplar (Populus trichocarpa Torr. & A.Gray ex Hook.) (ptc), and rice (Oryza sativa L.) (osa). Despite strong conservation of over 80%, some mature miR171 sequences, such as ptc-miR171j -l, and -m and osa-miR171g, -h, and -i, have undergone critical sequence variation, leading to functional diversification, since they target non-SCL gene transcript(s). Phylogenetic analyses revealed a combination of old ancestral relationships and recent lineage-specific diversification in the miR171 family within the four model plants. The cis-regulatory motifs on the upstream promoter sequences of miR171 genes were highly divergent and shared some similar elements, indicating their possible contribution to the functional variation observed within the miR171 family. This study will buttress our understanding of the functional differentiation of miRNAs and the relationships of miRNA-target pairs based on the evolutionary history of miRNA genes.

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.

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.

Strong electric field enhancement can be achieved in metal-insulator-metal (MIM) configuration where a thin dielectric layer is in between two metal layers. Here MIM nanostructures with intrinsic electromagnetic hot spots accessible for analytes are fabricated by oblique angle deposition (OAD). Firstly, silver nanoparticles are produced by OAD on a flat silver mirror with a thin dielectric over-layer. The broadband absorption peak of this particle-on-film system can be tuned by varying the particle size in order to fit a given excitation laser source for SERS (surface-enhanced Raman scattering). Then we deposit MIM nanoparticles on three-dimensionally structured silicon by an equivalent OAD method. By two-photon induced luminescence imaging and Raman measurements, we demonstrate strong and uniform field enhancement over the proposed substrate which is a good candidate for SERS application.

Simultaneous deep macroscopic imaging and microscopic imaging is in urgent demand, but is challenging to achieve experimentally due to the lack of proper fluorescent probes. Herein, we have designed and successfully synthesized simplex Er(3+)-doped upconversion nanoparticles (UCNPs) with double excitation bands for simultaneous deep macroscopic and microscopic imaging. The material structure and the excitation wavelength of Er(3+)-singly doped UCNPs were further optimized to enhance the upconversion emission efficiency. After optimization, we found that NaYF4:30%Er(3+)@NaYF4:2%Er(3+) could simultaneously achieve efficient two-photon excitation (2PE) macroscopic tissue imaging and three-photon excitation (3PE) deep microscopic when excited by 808 nm continuous wave (CW) and 1480 nm CW lasers, respectively. In vitro cell imaging and in vivo imaging have also been implemented to demonstrate the feasibility and potential of the proposed simplex Er(3+)-doped UCNPs as bioprobe.

UCNP-based drug delivery systems commonly rely on stimuli-sensitive auxiliaries, lacking a straightforward manipulation strategy. Here we designed Yb3+-enhanced upconversion/mesoporous silica nanocomposites (UCNP@SiO2) for consecutive cell imaging, photothermal drug delivery and cancer therapy. Core UCNPs (NaYbF4: 2% Er3+) were synthesized and coated with mesoporous silica, whose high-efficiency photothermal properties were verified in vitro. Then doxorubicin hydrochloride (DOX) was loaded on the UCNP@SiO2 and successfully triggered to release by a 975 nm laser of 150 mW or 300 mW. Before the therapy, we used a much lower laser power of 15 mW (which would cause little DOX release) for UCNP-probed fluorescence imaging of Hela cells and affirmed a favorable cell uptake of nanocomposites. Subsequently, cell viability assay and PI stain have demonstrated that the 300 mW laser could manipulate drug delivery of UCNP@SiO2-DOX and cause a severe loss of cell viability. The Yb3+-enhanced UCNP@SiO2 shows a great potential in simultaneous biomedical imaging and photothermal-triggered on-site drug delivery for chemotherapy of cancer.

Even though both dsRNA and siRNA have been widely used in laboratory studies to knock down target genes, it has been found that siRNA rarely triggers phenotypic changes for intact insects. In this study, Tribolium castaneum was used as a model species to compare dsRNA and siRNA and select the more efficient of these for insect RNAi.Four dsRNAs and ten siRNAs targeting the same three genes were tested by checking the suppression of target genes and the deformation of treated insects. The results showed that dsRNAs could reduce the expression of target genes by 55-75% for more than 7 days and deformed 78.8-87.0% of the larvae tested. Injection of their corresponding siRNAs targeting the same gene did yield about 45% silencing of target genes, but only in a short time period (from day 2 to day 4), and it did not trigger any phenotypic changes.SiRNAs were less efficient than their corresponding dsRNAs. Most of them could hardly trigger any phenotypic changes owing to the temporary silencing effect on target genes. Meanwhile, the efficiency of different siRNAs varied with their matching sequence, and careful selection was necessary for proper use.

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.

The solution space exponential explosion caused by the enumeration of the candidate solutions maybe is the biggest obstacle in DNA computing. In the paper, a new unenumerative DNA computing model for graph vertex coloring problem is presented based on two techniques: 1) ordering the vertex sequence for a given graph in such a way that any two consecutive labeled vertices i and i+1 should be adjacent in the graph as much as possible; 2) reducing the number of encodings representing colors according to the construture of the given graph. A graph with 12 vertices without triangles is solved and its initial solution space includes only 283 DNA strands, which is 0.0532 of 3(12) (the worst complexity).

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.

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands’ binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial “ASGPR” on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can’t bind to ASOR, it yet can’t form functional complex with ASGPRH2c.

AIM: To investigate the expressions of PTEN, PPM1A and P-Smad2 in hepatocellular carcinoma (HCC) and their significance. METHODS: The expressions of PTEN, PPM1A and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique. RESULTS: The positive expression (64.52%) and staining intensity (4.19 ± 3.31) of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues (97.37%, 7.88 ± 0.93; 100%, 7.77 ± 0.93, respectively) (P < 0.05), and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades II-III and above, was also lower than that of gradeIandI-II. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease (r = -0.339, P = 0.002); most of PPM1A might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson gradeIandI-II HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade ≥ II, weakly or negatively expressed in the nucleus (P < 0.05), and its location was negatively correlated with the progression of liver disease (r = -0.45, P = 0.0000). P-Smad2, which was mostly located in the nucleus and cytoplasm of gradeIandI-II HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade II and above (P < 0.001), and its location was positively correlated with the disease progression (r = 0.224, P = 0.016). Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPM1A (r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively); and PTEN and PPM1A were positively correlated with HCC carcinogenesis (r = 0.428, P = 0.000). CONCLUSION: The aberrant location of expression and staining intensity of PTEN, PPM1A and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

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.

399 PNPLA3 I148M AFFECTS LIVER STEATOSIS IN PATIENTS WITH CHRONIC HEPATITIS B M. Vigano’, L. Valenti, P. Lampertico, B.M. Motta, R. D’Ambrosio, F. Facchetti, P. Dongiovanni, S. Fargion, M. Colombo. Hepatology, Ospedale San Giuseppe IRCCS Multimedica, Internal Medicine, Universita’ di Milano, Fondazione IRCCS Ca’ Granda Milano, Gastroenterology, Fondazione IRCCS Ca’ Granda Milano, Universita degli Studi di Milano, Internal Medicine, Fondazione IRCCS Ca’ Granda Milano, Milan, Italy E-mail: luca.valenti@unimi.it

To clarify the relationship between the loss of expression of the deleted in pancreatic carcinoma locus 4 (DPC4) proteins and the pathogenesis of biliary tract carcinoma.71 primary biliary tract carcinoma (BTCa), including 38 common bile duct (CBD) carcinomas, 18 gallbladder carcinomas, 15 hilar bile ducts (HBD) carcinomas were examined by immunohistochemical staining. In addition, the CBD carcinomas were divided into two groups: tumors with metastasis (M(+) group, 27 cases) and tumors without metastasis (M(-) group, 11 cases).The frequency of loss of the expression of DPC4 protein was 32.8% in BTCa, 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, 13% in HBD carcinoma. Comparison of the frequency of loss expression of DPC4 was significant statistical difference in CBD carcinoma versus gallbladder carcinoma and HBD carcinoma (P0.01). The frequency of loss expression of DPC4 was 48.1% in the M(+) group and 45.4% in the M(-) group.There are a close relationship between pathogenesis of BTCa and inactivation of DPC4 and different frequencies of DPC4 gene alternation in various locations of the biliary tract, which are not significantly increased with tumor metastasis in BTCa.

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

Background and Aims: Pruritus is a common and disabling symptom in chronic cholestasis. The pathogenesis of this symptom is unknown but recently it has been associated with increased levels of lysophosphatidic acid. The role of other potential molecules is unknown, and therefore we have evaluated the serum metabolomic profile of patients with cholestatic pruritus. Methods: Serum samples were obtained from 36 female patients with primary biliary cirrhosis (PBC), 12 with severe pruritus, and 24 without pruritus, and from 10 healthy controls. All patients received ursodeoxycholic acid (13–15mg/kg/d) for the duration of the disease which was similar in the two groups (8.3±1.4 years). Metabolite extraction was accomplished by fractionating the serum samples into pools of species with similar physicochemical properties. All samples were randomized prior to the metabolite extraction procedure and three different profiles were used: a) polar lipids, non-esterified fatty acids, and bile acids; b) amino acids, and c) apolar lipids. The analyses were performed by UPLC-ESI-QTOFMS, random forests and multivariate data analysis. Results: Around 400 metabolites were identified in serum from patients with PBC. Patients with pruritus had significantly higher content of ceramides (p < 0.001), lysophosphatidylcholines (p = 0.03), phosphatidylcholines (p = 0.04), and triglycerides (p < 0.05) than patients without pruritus. Moreover, the detailed analysis of the lipid profile identified that patients with pruritus had higher levels of most lysophosphatidylcholines and phosphatidylcholines and lower contents of lysophosphatidylethanolamines and phosphatidylethanolamines. The serum level of ceramides, sphingomyelins and diacylglycerophosphoinositol was higher in patients with pruritus as well. No difference in the amino acid profile was observed between the two groups. The overall content of bile acids was not significantly different between patients with and without pruritus. However, a more detailed analysis observed higher amounts of glycocholic and taurocholic acid (p < 0.03), and lower levels of ursodeoxycholic and hyocholic acid (p < 0.05) in patients with pruritus. Conclusion: The metabolomic profile of patients with cholestatic pruritus is characterized by a marked change in the lipid metabolites, particularly in the ceramides, sphingomyelins and lysophospahtidylcholines. The analysis identified a panel of biomarkers that could effectively participate into the pathogenesis of pruritus. Parallel Session: PRE-CLINICAL HBV & HCV

after 2, 6, or 16 hours. Expression of CA markers (iNOS and TNF-a), AA markers (Fizz-1 and TGF-b), and matrix metalloproteinase-9 (MMP-9) were analyzed by real-time PCR. The role of b1-integrins was investigated by culturing in the presence or absence of integrin b1 blocking antibodies. Results: AA macrophages cultured on type III collagen significantly (P < 0.05) upregulated both the AA associated genes Fizz-1 and TGF-b and the CA associated genes TNF-a and iNOS; in contrast, those on type I collagen only reinforced the AA associated genes without effect on CA associated genes. Furthermore, AA macrophages on type III collagen significantly (P < 0.05) upregulated MMP-9, but this did not occur in any of the CA cultures. On type III collagen, TNF-a was maximally upregulated after 2 hours, whereas iNOS and MMP-9 showed the highest upregulation after 16 hours of culture. Inhibition of b1 integrin signalling resulted in downregulation of TNF-a, iNOS and MMP-9, although levels were still raised compared to culture on type I collagen. Conclusions: Type III collagen results in switching AA macrophages into an intermediate phenotype that has both AA and CA features, but also expresses MMP-9, which is neither a specifically described feature of CA or AA macrophages. The increased type III collagen production generally seen in early fibrosis may provide macrophages with such a phenotype in order to remove cellular and ECM debris. This phenotype is b1-integrin driven.

Background: The human asialoglycoprotein receptor (ASGPR) is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. Principal Findings: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR) composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using flourescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. Conclusions: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes. © 2010 Liu et al.

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.