Visualizing hepatitis C virus infections in human liver by two-photon microscopy.
OBJECTIVE
Although hepatitis C virus (HCV) is a common cause of cirrhosis and liver cancer, efforts to understand the pathogenesis of HCV infection have been limited by the low abundance of viral proteins expressed within the liver, which hinders the detection of infected cells in situ. This study evaluated the ability of advanced optical imaging techniques to determine the extent and distribution of HCV-infected cells within the liver.


METHODS
We combined 2-photon microscopy with virus-specific, fluorescent, semiconductor quantum dot probes to determine the proportion of hepatocytes that were infected with virus in frozen sections of liver tissue obtained from patients with chronic HCV infection.


RESULTS
Viral core and nonstructural protein 3 antigens were detected readily in liver tissues from patients with chronic infection without confounding tissue autofluorescence. Specificity was confirmed by blocking with specific antibodies and by tissue colocalization of distinct viral antigens. Between 7% and 20% of hepatocytes were infected in patients with plasma viral RNA loads of 10(5) IU/mL or greater. Infected cells were in clusters, which suggested spread of the virus from cell to cell. Double-stranded RNA, a product of viral replication, was abundant within cells at the center of such clusters, but often scarce in cells at the periphery, consistent with more recent infection of cells at the periphery.


CONCLUSIONS
Two-photon microscopy provides unprecedented sensitivity for the detection of HCV proteins and double-stranded RNA. Studies using this technology indicate that HCV infection is a dynamic process that involves a limited number of hepatocytes. HCV spread between cells is likely to be constrained by host responses.
Liang
Yuqiong
Y
Center for Hepatitis Research, Institute for Human Infections and Immunity, University of Texas Medical Branch at Galveston, Texas 77555-0610, USA.
Shilagard
Tuya
T
Xiao
Shu-Yuan
SY
Snyder
Ned
N
Lau
Daryl
D
Cicalese
Luca
L
Weiss
Heidi
H
Vargas
Gracie
G
Lemon
Stanley M
SM
D001706:Biopsy; D005260:Female; D016174:Hepacivirus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D006526:Hepatitis C / Q000175:diagnosis* / Q000473:pathology; D018936:Hepatitis C Antigens / Q000032:analysis*; D006801:Humans; D008099:Liver / Q000473:pathology / Q000821:virology*; D008297:Male; D036641:Microscopy, Fluorescence, Multiphoton; D008875:Middle Aged; D011237:Predictive Value of Tests; D045663:Quantum Dots; D012330:RNA, Double-Stranded / Q000032:analysis; D012367:RNA, Viral / Q000097:blood; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000032:analysis*; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000032:analysis*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018936:Hepatitis C Antigens; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases