Establishment Of A Novel HCV Infectious Cell Culture Model And Mouse Model

Hepatitis C virus (HCV) infection is a worldwide threat to the public health as ahigh risk of severe liver diseases, such as chronic liver hepatitis, liver cirrhosis andhepatocellular carcinoma. So far, lack of effective HCV vaccine increases thedifficulty in the control of HCV infection. As a result of the high species tropism ofHCV, the absence of a robust HCV infection system has severely limited the study ofHCV replication, pathogenesis, as well as the development of HCV vaccine andanti-HCV drugs. Thus, it is vital to establish a novel HCV infectious model,especially a small animal model, for both fundamental and applied research. In thisstudy, we took advantage of the Tet-on inducible system and the helper-dependentadenovirus (HDAd) system, respectively, for this purpose.1. Establishment of a Tet-on inducible uPA transgenic mouse model(1) Establishment of a Tet-on inducible uPA transgenic chimeric mouse modelThe human liver chimeric mouse model is generated through transplantation ofhuman hepatocytes into an immunodeficient mouse of which the mouse hepatocyteshave been undergoing programmed cell death, such as the Alb-uPA/SCID mousewhich is the first mouse model that supports HCV infection and evaluation ofanti-HCV compounds. Driven by the albumin promoter, the liver-specific constitutiveexpression of uPA causes murine liver injury which provides a microenvironment forthe engraftment of human primary hepatocytes. However, the high mortality rate atbirth, which ranges from70%to90%, makes it unstable and difficult to handle with.In this part, an inducible uPA transgenic chimeric mouse model was designed to bringdown the death rate. First of all, two transgenic constructs, namely Tet-on-Albuminand TRE2-uPA, were generated. The transcriptional activity of the Albumin promoter was tested and doxycycline (Dox)-induced expression of uPA was confirmed in vitro.Then Tet-on-Albumin and TRE2-uPA transgenic mice were generated throughpronuclear injection, respectively. The Tet-on-Albumin offsprings, in which rtTAspecifically expressed in the liver, were chosen and crossed with TRE2-uPAoffsprings to produce double transgenic Tet-on-Albumin/TRE2-uPA mice. AfterDox-induced expression of uPA in the liver of the double transgenic mouse wasconfirmed, the Tet-on-Albumin/TRE2-uPA/SCID mice were generated by crossingthe double trangenic mice with the immunodeficient SCID mice. Engraftment ofHepG2cells and Huh-7cells in the Tet-on-Albumin/TRE2-uPA/SCID mouse liverscould reach30%while the mortality rate was below5%. Thus, this inducible uPAtransgenic chimeric mouse model was successfully generated, which may further beapplied in the hepatitis C virus infection research.(2) HBV transfection and HCV infection research in the Tet-on inducible uPAtransgenic mouseTo investigate the relationship between over-expression of uPA and HBV relatedliver diseases, hydrodynamic transfection of plasmid adeno-associated virus(AAV)-1.3HBV was performed through the tail veins of the Dox-induced uPA doubletransgenic mice. Expression of uPA and HBV antigens were analyzed throughdouble-staining immunohistochemical assay. Cytokine production was detected byELISA. Results showed that plasmid AAV-1.3HBV hydrodynamic tranfection inDox-induced transgenic mice not only resulted in severe liver injury withhepatocarcinoma-like histological changes, but also showed an increased serum levelof HBV antigens and cytokines like interleukin-6and tumor necrosis factor-α,compared with the control group (p<0.01). Thus, over-expression of uPA plays asynergistic role in the development of liver injury, inflammation and regenerationduring acute HBV infection. Also, to investigate whether this inducible uPAtransgenic chimeric mouse model can be used in HCV infection research, we injectedthe HCV positive serum into the chiemric mouse through tail veins. Expression ofHCV RNA and proteins was undetectable. The reason may be that the infectious titreof the HCV serum was low. Further research will focus on the vector-mediatedtransfer of HCV genome. 2. Establishment of a HDAd-mediated HCV genomic cell culture model andmouse modelBeing immunodificient, the currently used human liver chimeric mouse modelcannot be used for the study of hepatitis viral immunopathogenesis and evaluation ofHCV vaccine. Thus, it is important to generate an immunocompetent mouse modelfor the above purposes. And it is a strategy to generate the hepatitis viral genomicanimal model through transgenic approaches or viral vector mediation. Among themostly used viral vectors, nearly none owns an enough capacity for the package ofnearly10kb HCV genome. However, by devoid of all the viral coding sequencesexcept the inverted terminal repeats (ITR) and packaging signal (Ψ), helper-dependentadenovirus (HDAd) has a large capacity of up to37kb. Also other advantages, such asthe high transduction efficiency, low toxicity and high hepatocyte tropism, make itsuitable for the package and delivery of HCV genome. This study for the first timeattempted to generate a HDAd-mediated HCV genomic replication system both invitro and in vivo, which may facilitate the study of HCV infection, pathogenesis, aswell as the development of HCV vaccine and therapeutic strategies.(1) Establishment of a HDAd-mediated HCV genomic cell culture modelSince recent years, several methods have been established and proven to beeffective in generating infectious HCV cell culture models–such as the invitro-transcribed JFH1-RNA transfection or stable transfection of the HCV cDNAinto human hepatoma Huh-7cells. However, other reliable and alternative methodsfor delivery of the HCV genome into cells are still worth trying. In this part, aHDAd-mediated HCV genomic cell culture model was generated as followes: HCVgenotype2a JFH1genome and a hepatitis delta virus (HDV) ribozyme sequence wereinserted into the HDAd vector. And recombinant HDAdJFH1virus was rescued,propagated in293Cre cells in the presence of a helper virus, and purified by CsClultracentrifugation, to reach a high titer. Then it was used to infect the human Huh-7hepatoma cells in vitro. Replication of the HCV genome was monitored by northernbot, western blot and immunofluorescence assay. And production of infectious HCVwas confirmed by infectivity neutralization experiment and electron microscopy. Ourresults demonstrate that infection of Huh-7cells with the HDAdJFH1virus led to efficient HCV replication and virion production. We found that the HCV viral RNAlevels could reach1.8×107copies per milliliter (ml) in the culture medium.HDAdJFH1-infected Huh-7cells could be cultured for8passages with the culturemedium remaining infectious for na ve Huh-7cells throughout this period. Thisinfection system also proved effective for evaluating the anti-HCV effect of IFN-α invitro. This system has also proven to be effective in generating infectious cell culturemodel for HCV genotype1b Con1wild-type genome.(2) Establishment of a HDAd-mediated HCV genomic mouse modelThe high species tropism of HCV to human and chimpanzees, render miceunsusceptible to HCV infection. Also the replication efficiency of HCV in mousehepatocytes is low. These issues urge the establishment of a novel HCV mouse modelfor HCV replication and infection. In this part, a HDAd-mediated HCV genomicmouse model was generated by a single injection of1×1010viral particles of purifiedHDAdJFH1virus into the tail veins of C57BL/6mice. Replication of HCV genomewas monitored by northern bot, western blot, and real-time quantative PCR. And thismouse model was used to evaluate the anti-HCV effect of LNA-antimiR-122, a novelanti-sense small molecular drug. Results suggested that in the HDAdJFH1infectedmouse livers, only transient expression of HCV proteins and genomic RNA wereobserved. Infectious hepatitis C virions were undetectable in the mouse liver andserum. However, this mouse model proved to be effective in evaluating the anti-HCVeffect of LNA-antimiR-122. Further work will focus on the humanization of mice forthe related studies.