Hepatitis C virus neuroinvasion: identification of infected cells

Abstract

Hepatitis C virus (HCV) infection often is associated with cognitive dysfunction and depression. HCV sequences and replicative forms were detected in autopsy brain tissue and cerebrospinal fluid from infected patients, suggesting direct neuroinvasion. However, the phenotype of cells harboring HCV in brain remains unclear. We studied autopsy brain tissue from 12 HCV-infected patients, 6 of whom were coinfected with human immunodeficiency virus. Cryostat sections of frontal cortex and subcortical white matter were stained with monoclonal antibodies specific for microglia/macrophages (CD68), oligodendrocytes (2',3'-cyclic nucleotide 3'-phosphodiesterase), astrocytes (glial fibrillary acidic protein [GFAP]), and neurons (neuronal-specific nuclear protein); separated by laser capture microscopy (LCM); and tested for the presence of positive- and negative-strand HCV RNA. Sections also were stained with antibodies to viral nonstructural protein 3 (NS3), separated by LCM, and phenotyped by real-time PCR. Finally, sections were double stained with antibodies specific for the cell phenotype and HCV NS3. HCV RNA was detected in CD68-positive cells in eight patients, and negative-strand HCV RNA, which is a viral replicative form, was found in three of these patients. HCV RNA also was found in astrocytes from three patients, but negative-strand RNA was not detected in these cells. In double immunostaining, 83 to 95% of cells positive for HCV NS3 also were CD68 positive, while 4 to 29% were GFAP positive. NS3-positive cells were negative for neuron and oligodendrocyte phenotypic markers. In conclusion, HCV infects brain microglia/macrophages and, to a lesser extent, astrocytes. Our findings could explain the biological basis of neurocognitive abnormalities in HCV infection.

FIG. 1.

LCM separation of macrophages/microglia, astrocytes, oligodendrocytes, neurons, and HCV-positive cells from HCV-positive brain tissue. Frontal cortex sections from patient 1 were stained with monoclonal antibodies against CD68, GFAP, CNPase, NeuN, and HCV NS3 protein. First column, stained tissue sections before LCM separation (subsequently separated cells are marked by arrows); middle column, sections after the positively staining cells were removed; last column, LCM-separated cells. The size of the bar corresponds to 150 nm.

FIG. 2.

Determination of the specificity of real-time RT-PCR for the phenotyping of brain cells (A to D) and determining the phenotype of HCV-infected cells in autopsy brain tissue (E and F). Frontal lobe tissue samples from patient 1 were stained with monoclonal antibodies specific for different cells phenotypes, separated by LCM, and subjected to four different RT-PCRs specific for astrocytes, microglia/macrophages, neurons, and oligodendrocytes. (A) GFAP-positive cells; (B) CD68⁺ cells; (C) NeuN-positive cells; (D) CNPase-positive cells. (E and F) NS3-positive cells for patients 3 and 4 were separated by LCM and analyzed by phenotype-specific RT-PCR. In both of these cases the CD68 transcript predominated.

FIG. 3.

Double immunocytochemical analysis of HCV-positive brains for markers of HCV infection and astrocytes and the macrophage/microglia phenotype. Frontal cortex sections from patient 3 were stained with monoclonal antibodies against HCV NS3 protein and against CD68 or GFAP. NS3 staining was visualized with DAB under normal light (first column), while phenotyping monoclonal antibodies were directly conjugated with fluorescein isothiocyanate (FITC) and visualized under UV light (second column). (A) Staining with anti-NS3 and anti-CD68 FITC-conjugated antibodies in an area in which virtually all NS3-positive cells also are CD68-positive (magnification, ×400). (B) The same staining in a different area, where most CD68-positive cells are NS3 negative. (C) Staining with anti-NS3 and anti-GFAP conjugated with FITC; arrows mark two cells that are positive for both markers (magnification, ×400). (D) The same stain in a different region; the NS3-positive cells all are negative for GFAP staining (magnification, ×200).