Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: A relevantex vivomodel of liver infection

Abstract

The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2-specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. Conclusion: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs. (HEPATOLOGY 2012;56:861-872) S tudies on the mechanisms of hepatitis C virus (HCV) infection have been hampered by limited in vivo and ex vivo models 1-4 as the consequence of poor replication of primary viral isolates and restriction of susceptible hosts (i.e., humans and chimpanzees). Recent advances have yielded cell-culture replication-competent chimeric viruses that have the capacity to achieve the complete infectious cycle. Of note, the infectious clone of genotype 2a HCV, Japanese fulminant hepatitis type 1 (JFH-1), 5,6 allowed us to develop chimeric viruses with exceptionally efficient replication, 7 because so-called cell-culture-grown hepatitis C virus (HCVcc) can be propagated, with infectivity titers in the range of 10 5 focus-forming units (ffu)/mL in the highly permissive Huh-7 subline, Huh-7.5.1. 8,9 However, this system has several limitations that include the inability to study the effects of pharmacologic inhibitors targeting the nonstructural proteins of the most prevalent, problematic viral strains (e.g., genotypes 1a and 1b). Moreover, the study of virus/host cell interactions is limited, because the permissive cell lines are transformed and poorly differentiated. Attempts to infect primary hepatocytes using primary HCV isolates resulted in limited poor viral replication and low production of de novo infectious virus particles. 10,11 Other models using primary human HCVcc-infected hepatocytes 12 or HCV-transfected hepatocytes cocultured with stellate cell lines 13 have been developed, which allowed virus replication of the entire viral life cycle with relatively high viral titers.

Cell Culture Systems for Hepatitis C Virus

“…A limitation of this study is still that this model is restricted to cell culture-derived viruses and it is unclear if also patient-derived viruses can be propagated in this system. A very recent study which is based on ex vivo human adult liver slices demonstrated a productive infection using human primary isolates of genotype 1b as well as JFH1 viruses and genotype 1 JFH1 chimeric genomes (Lagaye, Shen et al 2012). This new experimental model system in which viral titers above 10 5 ffu/ml were achieved allows in addition the validation of antiviral drugs.…”

Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway

“…It is still unclear whether discrepancies between models [9] could not also relate to active HCV replication in a non-physiological environment [76] , differences between viral genotypes [12] and/or differential processing of some HCV proteins [58] . Nevertheless, host factors involved in BHK-WNV cells were also required in Huh-7.5 cells that, over time, developed a cytoplasmic compartment [26] enriched in HCV core, CANX and RAB1.…”

“…hepatocytes [58] . In contrast, no clear effect of the inhibitor was observed on HCV production with BHK-WNV cells …”

Translational Research in Pharmacology and Toxicology Using Precision-Cut Tissue Slices

“…Recently it was shown that HCV infection could be studied in human liver slices. 168 For the rst time, the ability of primary HCV isolates to undergo de novo viral replication in human liver slices was demonstrated. This opens up the possibility of using liver slices to study virus infections that can lead to liver brosis.…”