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Gastroenterology. 2013 Dec;145(6):1404-13.e1-10. doi: 10.1053/j.gastro.2013.08.034. Epub 2013 Aug 22.

Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.

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  • 1Department of Medicine, Johns Hopkins University Baltimore, Maryland.

Abstract

BACKGROUND & AIMS:

Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples.

METHODS:

We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0-1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication.

RESULTS:

Patients' serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative.

CONCLUSIONS:

We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell-cell propagation of infection in the absence of clustered IFITM3.

Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

KEYWORDS:

AIDS Linked to Intravenous Experience; ALIVE; HCV; HIV; IFITM3; ISG; Intrahepatic Infection; LCM; PCR; Virology; cDNA; complementary DNA; hepatitis C virus; human immunodeficiency virus; interferon-induced transmembrane protein 3; interferon-stimulated gene; laser-capture microdissection; mRNA; messenger RNA; polymerase chain reaction; qPCR; quantitative polymerase chain reaction; scLCM; single-cell laser-capture microdissection; vRNA; viral RNA

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