Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver
Stefan F Wieland1, Zuzanna Makowska2, Benedetta Campana2, 3, Diego Calabrese2, Michael T. Dill2, 3, Josan Chung1, Francis V. Chisari1, Markus H. Heim2, 3
1Department of Molecular and Experimental Medicine, The Scripps Research Institute/ La Jolla, CA; 2Department of Biomedicine, University of Basel, Basel, Switzerland; 3Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland
Background: Over 20 years after the molecular cloning and identification of hepatitis C virus (HCV) a reproducible method to identify HCV infected hepatocytes in human liver biopsies is still lacking and this has been a major obstacle for understanding host-virus interactions in HCV infections. Methods: We adapted an in situ hybridization (ISH) system (QuantiGene® ViewRNA, Affymetrix, Santa Clara, CA) using HCV isolate specific probes. Snap frozen liver biopsies of 18 patients with chronic hepatitis C (CHC), different viral genotypes and a wide range of serum viral loads were analysed. For each biopsy, HCV RNA was isolated and sequenced, and highly specific probe sets were designed. We further developed the method using multiplex ISH to simultaneously detect HCV and interferon stimulated gene expression. Results: The system had an extremely high signal to noise ratio with virtually no unspecific background staining and no cross-reactivity by probe sets targeting plus or minus strand HCV RNA, different viral RNAs, and even between HCV isolates with identical genotypes, we found only weak cross-reactivity. The average percentage of infected hepatocytes in the 18 biopsies ranged from 1.3% to 53.9%. There was a significant positive correlation between the proportion of infected hepatocytes and the viral load in the serum and the liver, but not with the HCV genotype. HCV positive cells occurred in clusters. A quantitative analysis of the spatial relationship between HCV RNA and interferon stimulated gene (ISG) mRNA expression in the subset of patients with an induced endogenous IFN system revealed a significant correlation. ISG signal intensity was lowest in uninfected cells with uninfected neighbors, intermediate in uninfected cells with at least one HCV positive neighbor, and highest in HCV positive cells. Conclusion: Over 20 years after the cloning and identification of HCV, we have developed the first highly sensitive, specific and reproducible in situ detection systems that allows to identify HCV infected cells in liver biopsies in patients with viral loads as low as 1 0E4 IU/ml. A quantitative analysis of the number and spatial distribution of HCV infected cells and ISG expression revealed a number of fundamental question concerning HCV-host interactions: HCV infects only hepatocytes. The percentage of infected hepatocytes varies from 1 to 54 %, and correlates with serum viral load. HCV infected cells appear in clusters, favoring a model of cell to cell transmission. Finally, the positive correlation of HCV RNA signals with ISG mRNA expression in patients with induced ISG expression reveals that HCV is the central driver of ISG induction.
The following people have nothing to disclose: Stefan F Wieland, Zuzanna Makowska, Benedetta Campana, Diego Calabrese, Michael T. Dill, Josan Chung, Francis V. Chisari, Markus H. Heim