Mystery eye: Human adenovirus and the enigma of epidemic keratoconjunctivitis

Abstract

Known to occur in widespread outbreaks, epidemic keratoconjunctivitis (EKC) is a severe ocular surface infection with a strong historical association with human adenovirus (HAdV). While the conjunctival manifestations can vary from mild follicular conjunctivitis to hyper-acute, exudative conjunctivitis with formation of conjunctival membranes, EKC is distinct as the only form of adenovirus conjunctivitis in which the cornea is also involved, likely due to the specific corneal epithelial tropism of its causative viral agents. The initial development of a punctate or geographic epithelial keratitis may herald the later formation of stromal keratitis, and manifest as subepithelial infiltrates which often persist or recur for months to years after the acute infection has resolved. The chronic keratitis in EKC is associated with foreign body sensation, photophobia, glare, and reduced vision. However, over a century since the first clinical descriptions of EKC, and over 60 years since the first causative agent, human adenovirus type 8, was identified, our understanding of this disorder remains limited. This is underscored by a current lack of effective diagnostic tools and treatments. In part, stasis in our knowledge base has been encouraged by the continued acceptance, and indeed propagation of, inaccurate paradigms pertaining to disease etiology and pathogenesis, particularly with regard to mechanisms of innate and adaptive immunity within the cornea. Owing to its often persistent and medically refractory visual sequelae, reconsideration of key aspects of EKC disease biology is warranted to identify new treatment targets to curb its worldwide socioeconomic burden.

Keywords: Adaptive immunity; Epidemic keratoconjunctivitis; Human adenovirus; Innate immunity; Subepithelial infiltrates.

Fig. 1.

Ultrastructure and cross-sectional view of human adenovirus. (A) Schematic diagram of the icosahedral ultrastructure of HAdV, composed of20 triangular faces, 240 hexon trimers and 12 vertices. (B) Cross-sectional view through HAdV. (C) Corresponding table of important viral proteins. The HAdV capsid consists of major capsid proteins (hexon, penton, fiber knob and shaft) and minor capsid proteins (Ilia, VI, VIII, and IX). Capsid proteins encapsulate an assortment of core proteins, which include terminal DNA proteins, IVa2, Mu, V, VII and core proteases. The function and location of HAdV core proteins in particular remain a matter of speculation.

Fig. 2.

Overview of HAdV species, types, and associated clinical syndromes. As of the writing of this review, there are seven known species of HAdV (A-G) and 103 genotypes, the latter characterized as unique types either by the presence of novel coding sequences for at least one major capsid protein (penton, hexon, and fiber protein), or by a unique homologous recombination of the major capsid genes. The clinical manifestations of HAdV infection, with a focus on human adenoviral conjunctivitis, are also shown. Note types 91–103 were only recently published, characterized as type D adenoviruses. The sources of these HAdVs are unknown (Ismail et al., 2018a), and their disease associations have yet to be described.

Fig. 3.

Phylogenetic analysis of HAdV species and types. A phylogenetic tree of HAdV diversity performed for the first 60 types shows the proportional difference between genornes (Robinson et al., 2013a), reprinted with permission. There are now 103 HAdV types in GenBank.

Fig. 4.

Clinical manifestations of EKC. (A) Geographic epithelial keratitis manifesting as a large epithelial defect. (B) Fulminant membranous conjunctivitis with early symblepharon formation. (C) Slit lamp biomicroscopy of corneal SEIs seen in EKC. These are usually <0.5mm and relatively uniform, which may distinguish them from subepithelial keratitis seen in HSV and VZV keratitis in which SEIs tend to be larger and more varied in diameter.

Fig. 5.

Modeling formation of subepithelial infiltrates using the “human corneal facsimile”. (A) The 3-dimensional “corneal facsimile” consists of ① an overlying epithelial basement membrane mimic, Matrigel®, which contains the important molecule heparan sulfate;② primary human keratocytes distributed within a type I collagen matrix, and underlying porous supporting membrane. To model adenovirus keratitis, corneal keratocytes are infected with HAdV-D37 overnight, followed by the placement of human peripheral blood leukocytes in the media beneath the test tube insert, as seen in ③. (B) Remarkably, CD45+ leukocytes migrate against gravity through the matrix to fonn sub-basement membrane foci, similar to those seen in EKC. Localized chemotaxis to the sub-basement membrane is thought to occur due to co-localization of CXCL8 and heparan sulfate.

Fig. 6.

Confocal microscopy and histopathology of subepithelial infiltrate formation. (A) Confocal microscopy images of in vitro “corneal facsimiles”, with mock (M, top row) and HAdV-D37-infected facsimiles (V, bottom row), reproduced with permission (Rajaiya et al., 2015). Abundant CXCLS staining in virus-infected facsimiles, with CXCLS and heparan sulfate co-localization to the sub-basement membrane, are seen in the first and third columns, respectively. Staining with 4’, 6-diamidino-2-phenylindole (DAPI), as shown in the fourth column, demonstrated local infiltration of neutrophils to the sub-basement membrane area (white arrow). (B) Hematoxylin and eosin-stained histopathology of mock and infected facsimiles, showing a collection of neutrophils (black arrow) in the sub-basement membrane region only after virus infection.

Fig. 7.

Transmission electron microscopy of early changes in the C57Bl/6j mouse cornea following intra-stromal injection with 1μL (10⁵ tissue culture infectious doses) of HAdV-D37, reprinted with author permission (Mukherjee et al., 2015). Photomicrographs of stromal cells were taken (A) immediately, (B) 30 minutes, (C) 1 hour, and (D) 2 hours following infection. Cy – cytoplasm; Nu – nucleus; M – pericellular matrix. Depending on magnification, viral particles appear either as electron dense black dots. At 1 hour (C), the initial stages of viral entry is mediated by caevolae-dependent endocytosis (arrowhead), while cellular ruffling is seen in response to congregations of multiple viral particles (arrow). Within 2 hours (D), viral internalization within the stromal cell has occurred. Single viruses are internalized via endosomes (top arrowhead), while multiple viruses can be internalized via macropinosomes (bottom arrow). Magnification: (A) and (B) ×3400; (C) ×34000; and insets (A) and (B), and (D) ×19000. Scale bars: 2μm in (A) and (B), and 0.5μm in insets (A) and (B), and (C) and (D). The Association for Research in Vision and Ophthalmology retains copyright of this image.

Fig. 8.

Transmission electron microscopy of later changes in the C57Bl/6j mouse cornea following intra-stromal injection of 1μL (10⁵ tissue cultme infectious doses) of HAdV-D37, reprinted with author permission (Mukherjee et al., 2015). Photomicrographs at a magnification of × 3400 were taken at (A-C) 24 homs, (D) 48 homs and (E) 96 hours following infection. Ep – epithelium; N – neutrophil (polymorphonuclear cell); and K – keratocyte. The inflammatory response following infection is neutrophil-predominant, as shown by (A) stromal infiltration; (B) the beginning of phagocytosis; and (C) eventual phagocytosis. (D) shows whole viral particles (arrows) within stromal cells which now exist amidst aggregations of dense cellular debris. Such debris continues to accumulate and becomes increasingly evident at later time points (E). Scale bars: 2μm. The Association for Research in Vision and Ophthalmology retains copyright of this Image.

Fig. 9.

Adenoviral gene expression is not an absolute requirement for adenoviral keratitis. Molecular, cytological, macroscopic and histological evidence that adenoviral gene expression is not essential for the pathogenesis of keratitis, reprinted with author permission (Chintakuntlawar et al., 2010). In this experiment, mock (M), intact (V), UV-inactivated (UV), and heat-inactivated (H) HAdV-D37 was used to infect A549 cells in vitro and C57Bl/6j mouse corneas in vivo to determine if viral gene expression and replication were necessary to induce keratitis. (A) Relative expression of viral transcript ElAlOS in A549 cells, four hours following infection with HAdV-D37, as determined by real-time PCR. Intact virus (V) induced robust ElAlOS expression while mock (M), UV-inactivated (UV) and heat-inactivated (H) virus ElA expression was not detected. (B) To determine if inactivated virus could be internalized, in vivo confocal microscopy of C57Bl/6j mouse corneal stromal cells was performed 90 minutes following infection with Cy3-marked intact (V), UV-inactivated (UV), and heat-inactivated (H) virus. Intact (V) and UV-inactivated (UV) virus gained entry into cells, while heat-inactivated (H) virus did not. Cy3-labeled virus is shown in red; intracellular actin (phalloidin stain) in green; and nuclei (TO-PR03 stain) in blue. Scale: 20μm. (C) Photographs of C57Bl/6j mouse corneas post-infection. Within 24 hours, corneal opacification was demonstrated in mice infected with intact (V) and UV-inactivated (UV) virus. Infection with mock (M) and heat-inactivated (H) virus did not result in opacification. (D) Histopathology of representative mice corneas using hematoxylin and eosin, taken four days post-infection. Localized inflammation and stromal edema were observed in corneas infected with intact (V) and UV-inactivated (UV) virus. Negligible leukocyte infiltration was observed in corneas infected with a control buffer (M) or heat-inactivated (H) virus.

Fig. 10.

Synthesis of corneal stromal inflammatory responses to HAdV infection. This flowchart synthesizes our current understanding of the events which follow HAdV binding to corneal cells, with emphasis on the induction of pro-inflammatory pathways. In EKC-causing HAdV-D, a secondary interaction between HAdV RGD motifs and cellular integrins activate the Src-mediated signalosome, which is essential in viral internalization, prolongation of cell viability and therefore viral replication, and upregulation of inflammatory cytokine gene expression. The role of the humoral immune system in corneal pathogenesis is likely minor, but has not been investigated.