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Inferring viral dynamics in chronically HCV infected patients from the spatial distribution of infected hepatocytes.

Graw F, Balagopal A, Kandathil AJ, Ray SC, Thomas DL, Ribeiro RM, Perelson AS - PLoS Comput. Biol. (2014)

Bottom Line: We found that individual clusters on biopsy samples range in size from 4-50 infected cells.In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension.Further, we do not find a relationship between the cluster size and the estimated cluster expansion time.

View Article: PubMed Central - PubMed

Affiliation: Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, New Mexico, United States of America; Center for Modeling and Simulation in the Biosciences, Heidelberg University, Heidelberg, Germany.

ABSTRACT
Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.

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Relationship between cluster extension and HCV RNA content.(A) The maximal extension of the clusters, , characterized on the different grids in relation to the maximal cellular HCV RNA content in the cells belonging to this cluster. For each grid, only the clusters including the cell with the highest amount of intracellular HCV RNA were considered. Different patients are indicated by different symbols and colors. The radius  is given on a continuous scale, as well as converted into number of cells assuming radial spread. (B) The level of the HCV RNA content in hepatocytes with increasing cluster extension as characterized by Fig. 2. Dots correspond to the radii estimated during the iterative process of cluster determination. The individual results for all patients and all different sections per patient (grid 1 red, grid 2 blue, grid 3 green) are shown. Lines indicate the best fit of a model assuming a biphasic linear decrease of the intracellular HCV RNA content with increasing cluster extension (see Table 4). (C) The total HCV RNA content in an inferred cluster, i.e., the sum of the HCV RNA content in all hepatocytes belonging to a cluster versus the square root of the cluster size measured in number of cells. Each point is the result of 10,000 simulated clusters based on the characteristics for each of the different clusters per patient per grid as described in Materials & Methods. Symbols indicate the mean of total HCV RNA content and cluster size, arrows determine the 2.5% and 97.5% percentiles of the 10,000 bootstrap replicates. Corresponding values are given in Table 1.
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pcbi-1003934-g003: Relationship between cluster extension and HCV RNA content.(A) The maximal extension of the clusters, , characterized on the different grids in relation to the maximal cellular HCV RNA content in the cells belonging to this cluster. For each grid, only the clusters including the cell with the highest amount of intracellular HCV RNA were considered. Different patients are indicated by different symbols and colors. The radius is given on a continuous scale, as well as converted into number of cells assuming radial spread. (B) The level of the HCV RNA content in hepatocytes with increasing cluster extension as characterized by Fig. 2. Dots correspond to the radii estimated during the iterative process of cluster determination. The individual results for all patients and all different sections per patient (grid 1 red, grid 2 blue, grid 3 green) are shown. Lines indicate the best fit of a model assuming a biphasic linear decrease of the intracellular HCV RNA content with increasing cluster extension (see Table 4). (C) The total HCV RNA content in an inferred cluster, i.e., the sum of the HCV RNA content in all hepatocytes belonging to a cluster versus the square root of the cluster size measured in number of cells. Each point is the result of 10,000 simulated clusters based on the characteristics for each of the different clusters per patient per grid as described in Materials & Methods. Symbols indicate the mean of total HCV RNA content and cluster size, arrows determine the 2.5% and 97.5% percentiles of the 10,000 bootstrap replicates. Corresponding values are given in Table 1.

Mentions: After defining the clusters and their sizes, we analyzed the profile of the intracellular HCV RNA level within the clusters, i.e., the viral landscape or viroscape [15]. The relationship between the maximal radius of a cluster of infected cells and the HCV RNA content in the cell that presumably founded the cluster is shown in detail in Figure 3A. For example, the hepatocyte with the highest amount of HCV RNA on grid 3 of subject 2 contained 8.8 IU/cell, and the corresponding cluster of infected cells reached a maximal radius of (see Table 1). The mean cluster radius among all patients was (95%-CI [26.7, 57.9]) with variability between and within patients. There was no significant relationship between the maximal cluster radius and the HCV RNA content in the assumed founder cell of the cluster, i.e., the cell with the highest HCV RNA content (Figure 3A, linear mixed effects model). This result does not change if grids 1 of subject 1 and 3, which could be outliers (see above), are neglected.


Inferring viral dynamics in chronically HCV infected patients from the spatial distribution of infected hepatocytes.

Graw F, Balagopal A, Kandathil AJ, Ray SC, Thomas DL, Ribeiro RM, Perelson AS - PLoS Comput. Biol. (2014)

Relationship between cluster extension and HCV RNA content.(A) The maximal extension of the clusters, , characterized on the different grids in relation to the maximal cellular HCV RNA content in the cells belonging to this cluster. For each grid, only the clusters including the cell with the highest amount of intracellular HCV RNA were considered. Different patients are indicated by different symbols and colors. The radius  is given on a continuous scale, as well as converted into number of cells assuming radial spread. (B) The level of the HCV RNA content in hepatocytes with increasing cluster extension as characterized by Fig. 2. Dots correspond to the radii estimated during the iterative process of cluster determination. The individual results for all patients and all different sections per patient (grid 1 red, grid 2 blue, grid 3 green) are shown. Lines indicate the best fit of a model assuming a biphasic linear decrease of the intracellular HCV RNA content with increasing cluster extension (see Table 4). (C) The total HCV RNA content in an inferred cluster, i.e., the sum of the HCV RNA content in all hepatocytes belonging to a cluster versus the square root of the cluster size measured in number of cells. Each point is the result of 10,000 simulated clusters based on the characteristics for each of the different clusters per patient per grid as described in Materials & Methods. Symbols indicate the mean of total HCV RNA content and cluster size, arrows determine the 2.5% and 97.5% percentiles of the 10,000 bootstrap replicates. Corresponding values are given in Table 1.
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pcbi-1003934-g003: Relationship between cluster extension and HCV RNA content.(A) The maximal extension of the clusters, , characterized on the different grids in relation to the maximal cellular HCV RNA content in the cells belonging to this cluster. For each grid, only the clusters including the cell with the highest amount of intracellular HCV RNA were considered. Different patients are indicated by different symbols and colors. The radius is given on a continuous scale, as well as converted into number of cells assuming radial spread. (B) The level of the HCV RNA content in hepatocytes with increasing cluster extension as characterized by Fig. 2. Dots correspond to the radii estimated during the iterative process of cluster determination. The individual results for all patients and all different sections per patient (grid 1 red, grid 2 blue, grid 3 green) are shown. Lines indicate the best fit of a model assuming a biphasic linear decrease of the intracellular HCV RNA content with increasing cluster extension (see Table 4). (C) The total HCV RNA content in an inferred cluster, i.e., the sum of the HCV RNA content in all hepatocytes belonging to a cluster versus the square root of the cluster size measured in number of cells. Each point is the result of 10,000 simulated clusters based on the characteristics for each of the different clusters per patient per grid as described in Materials & Methods. Symbols indicate the mean of total HCV RNA content and cluster size, arrows determine the 2.5% and 97.5% percentiles of the 10,000 bootstrap replicates. Corresponding values are given in Table 1.
Mentions: After defining the clusters and their sizes, we analyzed the profile of the intracellular HCV RNA level within the clusters, i.e., the viral landscape or viroscape [15]. The relationship between the maximal radius of a cluster of infected cells and the HCV RNA content in the cell that presumably founded the cluster is shown in detail in Figure 3A. For example, the hepatocyte with the highest amount of HCV RNA on grid 3 of subject 2 contained 8.8 IU/cell, and the corresponding cluster of infected cells reached a maximal radius of (see Table 1). The mean cluster radius among all patients was (95%-CI [26.7, 57.9]) with variability between and within patients. There was no significant relationship between the maximal cluster radius and the HCV RNA content in the assumed founder cell of the cluster, i.e., the cell with the highest HCV RNA content (Figure 3A, linear mixed effects model). This result does not change if grids 1 of subject 1 and 3, which could be outliers (see above), are neglected.

Bottom Line: We found that individual clusters on biopsy samples range in size from 4-50 infected cells.In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension.Further, we do not find a relationship between the cluster size and the estimated cluster expansion time.

View Article: PubMed Central - PubMed

Affiliation: Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, New Mexico, United States of America; Center for Modeling and Simulation in the Biosciences, Heidelberg University, Heidelberg, Germany.

ABSTRACT
Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.

Show MeSH
Related in: MedlinePlus