title: Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver.
abstract: OBJECTIVE
  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.
authors: Department of Medicine, Johns Hopkins University Baltimore, Maryland.
authors: Abraham J
authors: AJ
authors: Kandathil
authors: Frederik
authors: F
authors: Graw
authors: Jeffrey
authors: J
authors: Quinn
authors: Hyon S
authors: HS
authors: Hwang
authors: Michael
authors: M
authors: Torbenson
authors: Alan S
authors: AS
authors: Perelson
authors: Stuart C
authors: SC
authors: Ray
authors: David L
authors: DL
authors: Thomas
authors: Ruy M
authors: RM
authors: Ribeiro
authors: Ashwin
authors: A
authors: Balagopal
mesh_terms: D000328:Adult; D001706:Biopsy; D003430:Cross-Sectional Studies; D005260:Female; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006526:Hepatitis C / Q000378:metabolism / Q000473:pathology*; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology* / Q000821:virology*; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology; D061346:Laser Capture Microdissection / Q000379:methods*; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008875:Middle Aged; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012189:Retrospective Studies; D015398:Signal Transduction / Q000502:physiology; D014779:Virus Replication
chemical_list: C080082:IFITM3 protein, human; D008565:Membrane Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins
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
references: Hepatology. 2002 May;35(5):1247-55
references: 11981775
references: PLoS Pathog. 2011 Oct;7(10):e1002337
references: 22046135
references: Cell. 1982 May;29(1):195-202
references: 6179628
references: J Virol. 2011 Jan;85(1):596-605
references: 20962076
references: Cell Host Microbe. 2012 Oct 18;12(4):558-70
references: 23084922
references: Proc Natl Acad Sci U S A. 2008 May 13;105(19):7034-9
references: 18467494
references: J Viral Hepat. 2011 Oct;18(10):e523-9
references: 21914072
references: Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7431-6
references: 20231459
references: Hepatology. 2008 Jan;47(1):17-24
references: 17941058
references: Hepatology. 1995 Mar;21(3):639-44
references: 7533121
references: Hepatology. 2012 Aug;56(2):544-54
references: 22331678
references: N Engl J Med. 1991 Jul 11;325(2):98-104
references: 1646962
references: Gastroenterology. 2009 Oct;137(4):1448-58
references: 19632233
references: J Virol. 2004 Dec;78(24):13779-92
references: 15564486
references: JAMA. 2000 Nov 22;284(20):2592
references: 11086358
references: J Virol. 2010 May;84(10):5067-77
references: 20200239
references: Science. 2000 Dec 8;290(5498):1972-4
references: 11110665
references: Gastroenterology. 2009 Jun;136(7):2377-86
references: 19303013
references: J Virol. 2006 Mar;80(5):2280-90
references: 16474135