Defining the spatial relationship between hepatitis C virus infection and interferon‐stimulated gene induction in the human liver

H epatitis C virus (HCV) is a hepatotropic RNA virus that chronically infects approximately 170 million people worldwide. HCV infection activates an interferon (IFN) response attributed to detection of HCV by various pattern recognition receptors in the infected liver. Though chronic HCV infection often results in high expression of hepatic IFN-stimulated gene (ISG) messenger RNAs (mRNAs), whose protein products are able to exert anti-HCV effects in cell-culture assays, the virus still persists. Why does this activated immune response fail to clear the virus in these patients? Though HCV infection does disrupt various host innate immune defenses, even within the infected liver, it is not clear what role this innate immune evasion plays during chronic HCV infection. Overall, the understanding of the spatial interplay of host immune activation in relationship to viral replication within the infected liver has been limited. In chronically infected livers, does HCV infection directly induce or prevent ISG expression in the infected hepatocyte or in neighboring cells? How does this induction of ISGs limit the spread of the virus within the liver (or not)? These questions can only be addressed by knowing which cells within the liver are infected with HCV, which cells express ISGs, and their spatial relationships during HCV infection. Detecting HCV infection within the liver has been challenging because of high autofluorescence and corresponding low signalto-noise ratios, as well as uncertain specificities of various staining approaches. However, in recent years, both immunostaining and in situ hybridization (ISH) approaches, as well as laser capture microdissection approaches combined with quantitative polymerase chain reaction, have been able to detect HCV RNA and proteins with increasing sensitivity. In this issue of HEPATOLOGY, Wieland et al. expand on these studies to address where interactions between HCV and the innate immune system occur within the chronically infected human liver. By using ISH with isolate sequence-specific probes in liver biopsies, the researchers were able to detect HCV positive-strand RNA signal specifically within hepatocytes, even in infected patients with very low viral loads ( 10 genomes/mL). The signal specificity was demonstrated by lack of background staining and no cross-reactivity of probe sets used with hepatitis B virus–infected livers or targeting either the positive or negative strands of HCV RNA. Additionally, there was little crossreactivity even between different sequence-specific probes targeted to identical genotypes. Positive HCV ISH signal was indicated by an HCV “signal dot,” and the number of signal dots per infected cell positively correlated with the proportion of infected cells per liver quadrant analyzed and with serum and liver viral loads, suggesting that the HCV signal dots are representative of authentic levels of HCV infection. The proportion of HCV-infected cells ranged from 1.3% to 54%, values consistent with other studies that quantified HCV infection within the liver. HCV-infected cells were found in clusters, in accord with others, supporting the idea that HCV infection occurs efficiently through cell-cell transfer. The clustered pattern of HCV infection suggests that virus spread could be restricted by local activation of ISG responses or, alternatively, that subsets of hepatocytes within the liver may have differing properties that permit or limit HCV infection or replication. The ultimate aim of the study by Wieland et al. was to identify the distribution of HCV-infected cells in relationship to the cells expressing ISG transcripts within the infected liver. For this, the researchers used a multiplex fluorescence ISH approach, combining Abbreviations: HCV, hepatitis C virus; IFN, interferon; ISG, IFN-stimulated gene; ISH, in situ hybridization; JAK, Janus kinase; mRNA, messenger RNA; STAT, signal transducer and activator of transcription. Address reprint requests to: Stacy M. Horner, Ph.D., Department of Molecular Genetics and Microbiology, Duke University Medical Center, 213 Research Drive, Box 3053 DUMC, Durham, NC 27710. E-mail: stacy.horner@duke.edu; fax: 919-613-8646. Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26960 Potential conflict of interest: Nothing to report.