Dynamics of hepatitis C virus replication in human liver

Abstract

Hepatitis C virus (HCV) replication at the cellular level is not fully understood. This study describes an optimized system for quantifying replication of HCV in hepatocytes and in liver tissues. A digital image analysis method was developed to quantify signal intensities of HCV genomic and replicative-intermediate RNAs in infected human liver tissues and to examine their spatial distribution. The average number of viral genomes per productively infected hepatocyte ranged from 7 to 64 RNA molecules. The maximal concentrations of genomic and replicative-intermediate RNAs at the single cell level were 74 and 34 molecules per hepatocyte, respectively. A gradient dispersion of genomes was observed around virus-producing cells, suggesting infection of neighboring hepatocytes as one mechanism of viral spread in the liver. There was no significant difference in total hepatic load of HCV genomes between the post- and nontransplant patients, whereas serum titers in the former group were much higher that that in the latter group. HCV replication varied among infected hepatocytes, occurred in a subset of cells, and proceeded at a low level, confirming one mechanism by which individual hepatocytes are cumulatively able to generate steady state concentrations of millions of HCV genomes per milliliter of blood. Lower viral clearance rates in circulating blood may explain the phenomenon of increased serum titers of viral RNA in posttransplant immunosuppressed patients.

Figure 1.

Fluorescent images of infected human liver tissue with HCV RNA signals. A and C: One HCV-infected liver tissue stained for genomic RNA and replicative-intermediate RNA, respectively. E: One non-HCV liver tissue probed with HCV anti-sense riboprobes. The HCV RNA-directed positive signals are shown in B and D. White arrows in A, C, and E point to nuclei of hepatocytes. A, C, and E are the superimposed pictures of DAPI, FITC, and rhodamine images. B, D, and F show rhodamine images of A, C, and E, respectively. Images were collected using the ×63 objective lens.

Figure 2.

Distribution of HCV genomic and negative strand RNA signal intensity values. Adjusted integrated signal intensities (AII) of all genomic or replicative-intermediate RNA signal particles in scanned images of each specimen are listed as log values. The signal particles were collected from three images of specimens As, Bs, and C; and five images of five cirrhotic nodules from specimens D and E, shown on the right.

Figure 3.

Fluorescent images of cell line Huh7-hcvAS with HCV RNA signals. A: A superimposed picture of Cy5, FITC, and rhodamine images. B: The rhodamine image containing signal particles of HCV subgenomic negative strand RNA. C: Cy5 image converted by the NIH Image program for calculating the number of nuclei.

Figure 4.

Gradient analysis. HCV genome density decreases as a function of distance from a single, very bright signal particle. The density of AII values (sum of AII values divided by area) was determined for a series of concentric circles drawn around the central particle. The inner and outer radii of these circles (0 to 25, 25 to 35, 35 to 43, 43 to 50, 50 to 56, and 56 to 78 μm, respectively) were set so that each ring would cover an area of ∼1963 square μm. The x axis gives the mean distance to the center for each ring. The y axis gives the sum of AII intensities within a ring divided by the area of the ring.