A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA.

BALB/c nude mice were injected intrahepatically with hepatitis B virus (HBV) DNA prepared from recombinant plasmids. Hepatitis B surface antigen appeared in the circulation in 19 of 23 mice (82%) 3 to 20 weeks postinjection and persisted for more than 6 months in most animals. Hepatitis B e antigen appeared transiently in the circulation in 12 of the 23 mice (52%) within a few weeks after the appearance of hepatitis B surface antigen. Antibodies to the core, X, and/or polymerase gene products of HBV have also been observed in 14 (61%) of the mice. Histopathological examination of the livers at 7 months postinjection demonstrated that nearly half had characteristics consistent with chronic hepatitis. HBV DNA appeared to be integrated into host liver DNA. No evidence of viral replication was observed in sera or livers from these mice at 7 months postinjection. These results demonstrate that an HBV chronic carrierlike state can be established in mice and that such a model could be used to study host and virus factors important in the establishment and maintenance of HBV-associated chronic liver disease.     

Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.     

siRNA Combinations Mediate Greater Suppression of Hepatitis B virus Replication in Mice

Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.     

Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.     

Synthesis and biological evaluation of nucleoside analogues than contain silatrane on the basis of the structure of acyclovir (ACV) as novel inhibitors of hepatitis B virus (HBV)

Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combination principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte was observed after treatment, as evaluated by flow cytometry (FCM).     

复制型HBV转基因小鼠遗传稳定性研究

目的：提高复制型HBV转基因小鼠的遗传稳定性。方法：应用回交传代及双杂交育种法,经荧光定量PCR、ELISA和化学发光法研究HBV基因在小鼠体内的复制与表达。结果：HBV转基因小鼠已稳定传至第23代,血清HBsAg达4122.31±2044.74IU/ml,93.93%的转基因小鼠血清HBV DNA达104-106copies/ml,表达复制水平较早期有显著提高并稳定传代;雌雄小鼠之间表达水平无显著性差异。结论：该转基因小鼠经过培育传代,已成为一个高表达且遗传稳定的复制型HBV小鼠模型。     

Mechanism of random integration of foreign DNA in transgenic mice

Little is known about how foreign DNA is randomly integrated into chromosomes in transgenic animals. In the current study, the insertion sites of 36 transgenic mice were mapped by thermal asymmetric interlaced PCR, and 38 junction sequences were obtained from 30 samples. Analysis of the 38 sequences revealed that 44.7 % of integration events occurred within host gene regions, including 13.2 % (5/38) in exonic regions and 31.6 % (12/38) in intronic regions. The results also revealed that all non-end side integrations of foreign DNA were mediated by short sequence homologies (microhomologies) and that the end side integrations occurred in the presence or absence of microhomologies. In addition, microhomology-mediated mechanisms were also confirmed in four transgenic Arabidopsis thaliana lines. The results indicate that foreign DNA is easily integrated into host gene regions. These results also suggest that the integration of both ends of foreign DNA follows the above-mentioned mechanism in many transgenic/transformed organisms.     

Tumor Necrosis Factor-Alpha Induced by Hepatitis B Virus Core Mediating the Immune Response for Hepatitis B Viral Clearance in Mice Model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8⁺ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.     

大剂量HBsAg对HBV转基因小鼠T细胞免疫调节的研究

目的：研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法：用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果：HBsAg组免疫后脾细胞产生的Th1类细胞因子（IFN-γ、IL-2）、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加（P〈0.05）。结论：大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

Autophagy required for hepatitis B virus replication in transgenic mice

Recent studies indicate that hepatitis B virus (HBV) may induce autophagy to enhance its replication in cell cultures. To understand whether autophagy can indeed enhance HBV replication in vivo, we generated HBV transgenic mice with liver-specific knockout of the Atg5 gene, a gene critical for the initiation of autophagy. Immunoblot analyses confirmed the inhibition of autophagy in the livers of Atg5 knockout mice. This inhibition of autophagy slightly reduced HBV gene expression and affected nuclear localization of the HBV core protein. It also reduced the HBV DNA level in sera by more than 90% and the level of the HBV DNA replicative intermediate in the mouse liver to an almost undetectable level. Our results thus demonstrate that autophagy is important for HBV replication in vivo and raise the possibility of targeting this pathway to treat HBV patients.     

Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus

Inhibition of hepatitis B virus (HBV) replication and viral clearance from an infected host requires both the innate and adaptive immune responses. Expression of interferon (IFN)-inducible proteasome catalytic and regulatory subunits correlates with the IFN-alpha/beta- and IFN-gamma-mediated noncytopathic inhibition of HBV in transgenic mice and hepatocytes, as well as with clearance of the virus in acutely infected chimpanzees. The immunoproteasome catalytic subunits LMP2 and LMP7 alter proteasome specificity and influence the pool of peptides available for presentation by major histocompatibility complex class I molecules. We found that these subunits influenced both the magnitude and specificity of the CD8 T-cell response to the HBV polymerase and envelope proteins in immunized HLA-A2-transgenic mice. We also examined the role of LMP2 and LMP7 in the IFN-alpha/beta- and IFN-gamma-mediated inhibition of virus replication using HBV transgenic mice and found that they do not play a direct role in this process. These results demonstrate the ability of the IFN-induced proteasome catalytic subunits to shape the HBV-specific CD8 T-cell response and thus potentially influence the progression of infection to acute or chronic disease. In addition, these studies identify a potential key role for IFN in regulating the adaptive immune response to HBV through alterations in viral antigen processing.     

Interferon inhibits hepatitis B virus replication in a stable expression system of transfected viral DNA.

The effect of interferon (IFN) on hepatitis B virus (HBV) replication was investigated in a stable expression system, using HepG2 cells transfected with recombinant HBV DNA. IFN was found to cause a marked reduction in the levels of both minus and plus strands of HBV DNA from core particles in the cytoplasm. Neither HBV DNA from virus particles nor the HBV surface antigen in the culture medium primarily underwent change in quantity by treatment with IFN, as was also found for HBV mRNAs and the HBV core antigen/HBV e antigen in the cytoplasm. IFN exerted no influence on HBV DNA synthesis by endogenous DNA polymerase in the core particle fraction. From these findings, it would appear that IFN inhibits HBV replication by blocking some step in the pregenome RNA-primed assembly of core particles.