Advances and challenges in studying hepatitis C virus in its native environment.
ABSTRACT Approximately 2% of the worldwide population is infected with hepatitis C virus (HCV), the major causative agent of non-A, non-B hepatitis. Although substantial progress has been made in developing tools to dissect the viral life cycle, most in vitro studies rely on hepatoma cell lines, which are functionally disparate from the natural in vivo target of the virus – hepatocytes. To gain insights into virus–host interactions, there is a need for HCV-model systems that more closely mimic the physiological environment of the liver. Here, we discuss recent advances in culture and detection systems that facilitate the study of HCV in primary cells. Use of these new models may help bridge the gap between in vitro studies and clinical research.
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Article: HCV animal models and liver disease[Show abstract] [Hide abstract]
ABSTRACT: The development and evaluation of effective therapies and vaccines for the hepatitis C virus (HCV) and the study of its interactions with the mammalian host have been hindered for a long time by the absence of suitable small animal models. Due to the narrow host tropism of HCV, the development of mice that can be robustly engrafted with human hepatocytes was a major breakthrough since they recapitulate the complete HCV life cycle. This model has been useful to investigate many aspects of the HCV life cycle, including antiviral interventions. However, studies of cellular immunity, immunopathogenesis and resulting liver diseases have been hampered by the lack of a small animal model with a functional immune system. In this review, we summarize the evolution of in vivo models for the study of HCV.Journal of Hepatology 11/2014; 61(1). DOI:10.1016/j.jhep.2014.07.013 · 10.40 Impact Factor
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ABSTRACT: It is well known that NS3/4A protein plays crucial roles in the hepatitis C virus (HCV) replication. NS3/4A protein also results to virus-mediated immune evasion and persistence of infection through the interaction with host proteins. However, the lack of a suitable animal model hampers studies of HCV NS3/4A protein interaction with host proteins, which impacts immunopathology due to infection. Here, transgenic vector containing transcriptional regulation and Fluc reporter gene was constructed to conditionally express NS3/4A protein under the dual control of Tet-On regulatory system and Cre/LoxP gene-knockout system. NS3/4A transgenic founder mice were continuously crossed with Lap transgenic mice expressing reverse tetracycline-controlled transcriptional activator (rtTA), the NS3/4A/Lap double transgenic mouse lines with liver-specifically and conditionally expressing reporter (luciferase Fluc) under control of Tet-On system were established. The NS3/4A/Lap double transgenic mouse are mated with Lap/LC-1 double transgenic mouse with liver-specifically and conditionally expressing Cre recombinase under control of Tet-On system, NS3/4A/Lap/LC-1 triple transgenic mouse were generated. In vivo bioluminescent imaging, western blotting and immunohistochemical staining (IHS) was used to confirm that NS3/4A protein was strictly expressed in the liver of Doxycycline-induced triple transgenic mice. The results show that we established a triple-transgenic mouse model conditionally expressing the HCV NS3/4A protein under strict control of the Tet-On regulatory system and Cre/loxP system. This novel transgenic mouse model expressing NS3/4A in a temporally and spatially-specific manner will be useful for studying interactions between HCV NS3/4A protein and the host, also for evaluating NS3/4A protease inhibitors.Molecular Biology Reports 09/2014; 41(11). DOI:10.1007/s11033-014-3621-8 · 1.96 Impact Factor
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ABSTRACT: Primary human hepatocytes are considered the ideal cellular model for in-vitro studies of liver-specific pathology, such as hepatitis B virus (HBV) infection. However, poor accessibility, limited cell numbers, and lot-to-lot variation of primary human hepatocytes limit their broad application. Human fetal hepatocytes were isolated from postmortem embryonic liver tissues by two-step collagenase perfusion and cryopreserved. A monolayer of cryopreserved human fetal hepatocytes was established by optimizing such conditions as cell density and viability and purification of viable cells by Percoll. Finally, revived human fetal hepatocytes were characterized and infected with HBV. A large number of viable human fetal hepatocytes could be isolated and cryopreserved, with seeding density and viability being critical for the establishment of a compact monolayer culture. Using low-viability cryopreserved human fetal hepatocytes, a typical monolayer was established by purification with Percoll. The revived cells were actively proliferative, showed identical morphologic characteristics to non-cryopreserved cells, and had a typical hepatic gene expression profile. Moreover, this optimized model was susceptible to HBV infection and could be used to screen entry inhibitors against HBV infection. In conclusion, these methods can be used on human fetal hepatocytes to provide a cell bank for studies of the early stages of HBV infection.Journal of Virological Methods 06/2014; 207. DOI:10.1016/j.jviromet.2014.06.015 · 1.88 Impact Factor