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. 2017 Jul 14;6(4):12.
doi: 10.1167/tvst.6.4.12. eCollection 2017 Jul.

Retinal Pigment Epithelial Cells Are a Potential Reservoir for Ebola Virus in the Human Eye

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Free PMC article

Retinal Pigment Epithelial Cells Are a Potential Reservoir for Ebola Virus in the Human Eye

Justine R Smith et al. Transl Vis Sci Technol. .
Free PMC article

Abstract

Purpose: Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells.

Methods: We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction.

Results: Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege.

Conclusions: Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye.

Translational relevance: This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV.

Keywords: Ebola virus; eye; human; infection; pigment epithelial cell.

Figures

Figure 1
Figure 1
Infection of human retinal pigment epithelial cells with EBOV (multiplicity of infection = 5; evaluated time-points post inoculation = 4, 24, 48, and 72 hours). (A) EBOV-infected and mock-infected ARPE-19 cells immunolabeled to detect Ebolavirus nucleoprotein. Alexa Fluor 488 (green) with DAPI nuclear counterstain (blue). Original magnification: ×10. (B) Graph of TCID50 for culture supernatant collected from EBOV-infected ARPE-19 cell monolayers. n = 3 cultures/condition. Dots represent mean TCID50/mL, with error bars showing SD. (C, D) Graphs showing relative transcript expression for selected viral RNA pattern recognition receptors (C) and inflammatory molecules (D) in EBOV-infected ARPE-19 cells versus mock-infected cells. Bars represent mean relative expression, with error bars showing SEM. n =3 cultures/condition, with exception of 72-hour mock-infected (2 cultures, pooled, and tested in triplicate). Data were analyzed by two-tailed Student's t-test.
Figure 2
Figure 2
Gene expression changes in human retinal pigment epithelial cells infected with EBOV (multiplicity of infection = 5; evaluated time-point post inoculation = 24 hours) by RNA sequencing. (A, B) Graphs present 10 most highly enriched pathways (A) and gene ontology terms (B) for the 560 gene products that were significantly more highly expressed in EBOV-infected ARPE-19 cells in comparison to mock-infected cells, as annotated in InnateDB. Horizontal bars indicate –log10 false discovery rate for each pathway or gene ontology term. (C) Heatmap of normalized counts per million for InnateDB-annotated IFN-regulated genes. Color scale runs from yellow to red, with higher intensity red representing higher counts for each transcript in either the mock-infected or EBOV-infected ARPE-19 cells. n = 3 cultures/condition. USP18, ubiquitin specific peptidase 18; OAS, 2′,5′-oligoadenylate synthetase; MX1, MX dynamin-like GTPase 1; ISG, IFN-stimulated gene; IFITM, IFN-induced transmembrane protein; IFIT, IFN-induced protein with tetratricopeptide repeats; IFI6, IFN-α inducible protein 6; EGR1, early growth response 1.
Figure 3
Figure 3
Network analysis of gene expression changes in human retinal pigment epithelial cells infected with EBOV (multiplicity of infection = 5; evaluated time-point post inoculation = 24 hours) by RNA sequencing. (A) Heatmap of the normalized counts per million for genes that were differentially expressed between EBOV-infected or mock-infected ARPE-19 cells. n = 3 cultures/condition. Color scale runs from yellow to red, with higher intensity red representing higher counts for each transcript. (B–D) Network schematics representing interactions between differentially expressed genes and their first neighbor interactors, as annotated in InnateDB. Lines indicate molecular interactions; red nodes indicate upregulated transcripts; green nodes indicate downregulated transcripts; pink nodes indicate interactors that are not differentially expressed. Node size is proportional to fold-change in gene expression. (C) High-scoring subnetwork enriched in upregulated transcripts. (D) High-scoring subnetwork enriched in downregulated transcripts. Enlarged versions of (C, D) are presented as Supplementary Figs. S2 and S3.
Figure 4
Figure 4
Anti-viral type I IFN response of human retinal pigment epithelial cells infected with EBOV (multiplicity of infection = 5; evaluated time-points post inoculation = 4, 24, 48, and 72 hours). Graphs showing relative transcript expression for IFN-α and IFN-β, plus the IFN receptor subunits, IFN-αR1 and IFN-αR1, and selected IFN-stimulated gene products in EBOV-infected ARPE-19 cells versus mock-infected cells. Reference genes were β-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing SEM. n =3 cultures/condition, with exception of 72-hour mock-infected (2 cultures, pooled, and tested in triplicate). Data were analyzed by two-tailed Student's t-test. EIF2AK2 = eukaryotic translation initiation factor 2-alpha kinase 2; RSAD2 = radical SAM domain-containing 2; BST2 = bone marrow stromal antigen 2.
Figure 5
Figure 5
Expression of immunomodulatory molecules by human retinal pigment epithelial cells infected with EBOV (multiplicity of infection = 5; evaluated time-points post inoculation = 4, 24, 48 and 72 hours). Graphs showing relative transcript expression for selected immunomodulatory molecules in EBOV-infected ARPE-19 cells versus mock-infected cells. Reference genes were β-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, and TATA-binding protein. Bars represent mean relative expression, with error bars showing SEM. n =3 cultures/condition, with exception of 72 hour mock-infected (2 cultures, pooled, and tested in triplicate). Data were analyzed by two-tailed Student's t-test.

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