Use of Laser Capture Microdissection to Map Hepatitis C Virus–Positive Hepatocytes in Human Liver

Kandathil, AJ; Graw, Frederik; Quinn, Jeffrey; Hwang, Hyon S.; Torbenson, Michael; Perelson, Alan S.; Ray, Stuart C.; Thomas, David L.; Ribeiro, Ruy M.; Balagopal, Ashwin

doi:10.1053/j.gastro.2013.08.034

Cited by 76 publications

(129 citation statements)

References 23 publications

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“…2B). Laser capture microdissection was used to enrich hepatocytes from liver tissue collected from subjects ISV-1 and ISV-2, as previously described (24): RNA was extracted and studied by NGMS. HHpgV-1 reads were identified in the hepatocytes of subject ISV-1 but not from subject ISV-2.…”

Section: Resultsmentioning

confidence: 99%

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

et al. 2017

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Background Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings. Objective To explore the plasma virome of persons who inject drugs while characterizing the sensitivity and accuracy of NGMS compared to quantitative clinical standards. Design Longitudinal and cross-sectional studies. Participants Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infected persons enrolled in a clinical trial (NCT01285050) and/or a well-characterized cohort study of persons who have injected drugs. Measurements Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR). Results NGMS generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained >10,000 copies or IU/ml as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1) was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Because of this unexpected finding, using a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs in whom HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses such as HCV RNA and SEN virus D Limitations The medical importance of HHpgV-1infection remains unknown. Conclusions Although NGMS is insensitive to detection of viruses with relatively low plasma nucleic acid concentrations, it may have a broad potential for discovery of new viral infections of potential medical importance like HHpgV-1.

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Section: Resultsmentioning

confidence: 99%

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

et al. 2017

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“…Previous studies performed by in situ PCR (33,34), fluorescence microscopy (35), or laser-capture microdissection followed by RT-PCR (36,37) indicated that the distribution of HCV RNA in the liver is focal and that the percentage of infected hepatocytes is low (never >35% of the total population). However, the focal distribution of HCV-infected cells (37) cannot explain the restricted viral replication that we detected exclusively within the tumor.…”

Section: Discussionmentioning

confidence: 99%

Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

Harouaka

Engle

Wollenberg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.hepatitis C virus | hepatocellular carcinoma | HCV RNA levels | HCV quasispecies | liver H epatitis C virus (HCV) is a hepatotropic, single-stranded RNA virus that replicates in the cytoplasm of hepatocytes and does not integrate into the host genome (1). HCV circulates in vivo as a dynamic distribution of closely related viral variants that are commonly referred to as "quasispecies" (2). Such diversity confers a remarkable advantage to the virus under host selective constraints (3-5). One of the most important features of HCV is its extraordinary ability to persist in up to 80% of infected individuals (6). Approximately 20-30% of chronic HCV carriers develop cirrhosis and its long-term sequelae, including hepatic decompensation and hepatocellular carcinoma (HCC), leading to orthotopic liver transplantation (OLT) or liver-related death (6). The incidence of HCC in the United States has more than tripled over the past 3 decades (7), an alarming trend due primarily, if not exclusively, to HCV infection (8).Although epidemiologic evidence has linked chronic HCV infection to a significantly elevated risk of developing HCC (9), the mechanisms whereby HCV promotes hepatocarcinogenesis remain to be elucidated (10). Whether HCV elicits liver cancer indirectly, through chronic inflammation, fibrosis, and continuous liver regeneration (11), or directly, through the expression of tumor-promoting viral proteins, in a manner analogous to other oncogenic viruses, such as human papillomaviruses and EpsteinBarr virus (12, 13), remains unknown. The major challenges in defining the role of HCV in the pathogenesis of HCC include the inherent limitations of available experimental...

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“…While we cannot absolutely exclude such a possibility, it seems unlikely for several reasons. First, multiple modalities, including two-photon fluorescence microscopy (2), fluorescent in situ RNA hybridization (31), and single-cell laser capture microdissection coupled with qRT-PCR (32), all indicate that HCV replicates very inefficiently with limited expression of viral proteins and RNA within individual hepatocytes in the infected human liver. Infected hepatocytes are estimated to contain no more than 100 copies of the HCV genome (33), far fewer than JFH1 (or even H77S.3) virus-infected Huh-7.5 cells.…”

Section: Discussionmentioning

confidence: 99%

Dissecting the Roles of the 5′ Exoribonucleases Xrn1 and Xrn2 in Restricting Hepatitis C Virus Replication

Yamane

Lemon

2015

J Virol

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The replication of hepatitis C virus (HCV) is uniquely dependent on a host microRNA, miR-122. Previous studies using genotype 1a H77S.3 virus demonstrated that miR-122 acts in part by protecting the RNA genome from 5= decay mediated by the cytoplasmic 5= exoribonuclease, Xrn1. However, this finding has been challenged by a recent report suggesting that a predominantly nuclear exoribonuclease, Xrn2, mediates the degradation of genotype 2a JFH1 RNA. Here, we dissect the roles of these two 5= exoribonucleases in restricting the replication of different HCV strains and mediating the decay of HCV RNA. Small interfering RNA (siRNA) depletion experiments indicated that Xrn1 restricts replication of all HCV strains tested: JFH1, H77S.3, H77D (a robustly replicating genotype 1a variant), and HJ3-5 (a genotype 1a/2a chimeric virus). In contrast, the antiviral effects of Xrn2 were limited to JFH1 and H77D viruses. Moreover, such effects were not apparent in cells infected with a JFH1 luciferase reporter virus. Whereas Xrn1 depletion significantly slowed decay of JFH1 and HJ3-5 RNAs, Xrn2 depletion marginally enhanced the JFH1 RNA half-life and had no effect on HJ3-5 RNA decay. The positive effects of Xrn1 depletion on JFH1 replication were largely redundant and nonadditive with those of exogenous miR-122 supplementation, whereas Xrn2 depletion acted additively and thus independently of miR-122. We conclude that Xrn1 is the dominant 5= exoribonuclease mediating decay of HCV RNA and that miR-122 provides protection against it. The restriction of JFH1 and H77D replication by Xrn2 is likely indirect in nature and possibly linked to cytopathic effects of these robustly replicating viruses. IMPORTANCEHCV is a common cause of liver disease both within and outside the United States. Its replication is dependent upon a small, liver-specific noncoding RNA, miR-122. Although this requirement has been exploited for the development of an anti-miR-122 antagomir as a host-targeting antiviral, the molecular mechanisms underpinning the host factor activity of miR-122 remain incompletely defined. Conflicting reports suggest miR-122 protects the viral RNA against decay mediated by distinct cellular 5= exoribonucleases, Xrn1 and Xrn2. Here, we compare the roles of these two exoribonucleases in HCV-infected cells and confirm that Xrn1, not Xrn2, is primarily responsible for decay of RNA in cells infected with multiple virus strains. Our results clarify previously published research and add to the current understanding of the host factor requirement for miR-122. P ersistent infections with hepatitis C virus (HCV) are a common cause of chronic and life-threatening liver diseases, including cirrhosis and hepatocellular carcinoma (1). A positivestrand RNA virus classified within the genus Hepacivirus in the family Flaviviridae, HCV typically infects surreptitiously, causing little overt disease until late in the course of the infection, often several decades later. The virus is largely, if not exclusively, hepatotropic, but viral RNA and p...

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Use of Laser Capture Microdissection to Map Hepatitis C Virus–Positive Hepatocytes in Human Liver

Cited by 76 publications

References 23 publications

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

Dissecting the Roles of the 5′ Exoribonucleases Xrn1 and Xrn2 in Restricting Hepatitis C Virus Replication

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