Herpes simplex virus type-1 infection and spread in a novel porcine corneal explant model is restricted to the epithelium

Abstract

Herpes Keratitis (HK) is a debilitating infection of the cornea that remains the leading cause of infectious blindness in developed countries. Caused primarily by herpes simplex virus type 1 (HSV-1), it is associated with recurrent inflammation, leading to corneal scarring. This study investigated the initial events during acute HSV-1 infection in the cornea by adapting our human anogenital mucosal explant model to a HSV-1 infected porcine corneal explant model. We infected these corneas topically via high-density microarray patches (HD-MAPs) dipped in GFP-labelled HSV-1. Virus infection and spread was detected by both GFP protein and RNAscope, adapted for HSV-1 DNA. The punctures were consistent, usually in the epithelium but some extended into the underlying stroma. However, HSV-1 was restricted to the corneal epithelium, without spread through the anterior limiting membrane (ALM) or Bowman's layer into the stroma nor to the uppermost epithelial layer. This layer expressed SPRR1A similarly to the stratum granulosum of skin which is refractory to HSV-1 infection. In corneas where infected epithelial cells extended to the ALM, SPRR1A was also observed in this layer, suggesting it may contribute to its barrier function. Such studies of HSV-1 infection and spread will help improve therapy for HK and vaccine design to prevent it.

Fig 1. Development of a novel corneal explant model to investigate the spread of acute HSV-1 infection.

(A) Histology of porcine and human cornea. Ep = epithelium, ALM = anterior limiting membrane, S = stroma, PLM = posterior limiting membrane, En = endothelium. (B) Set-up for the excision of the cornea from the porcine orbital globe. (C) Porcine corneas were subjected to HSV-1-treated (1 x 10⁸ PFU/mL) HD-MAPs or mock-treated HD-MAPs and cultured for 24 hours at 37˚C. Tissue was processed for RNAscope and IF staining. (D) Protease Plus digestion of 10 minutes for RNAscope was optimal in the porcine cornea for detection of positive control probe for Cyclophilin B RNA (red). (E) HSV-1-pre-treated HD-MAPs resulted in detectable infection of HSV-1-GFP, labelled using rabbit anti-GFP (1:1000) primary and donkey anti-rabbit AF488 (1:400) secondary (green) antibodies within the porcine cornea, whereas topical infection via cloning cylinders did not. Arrows = punctures. Scale bars = 50μm or as indicated. Schematic diagrams created in MS PowerPoint and BioRender.com.

Fig 2. Detection of HSV-1 DNA via GFP and RNAscope in porcine corneal epithelium.

Punctures in the patch-treated porcine corneal epithelium mostly healed by 24 hours but some penetrated into the stroma and were still obvious at this time. (B) Porcine corneas were subjected to HSV-1-treated (1 x 10⁸ PFU/mL) or mock treated HD-MAPs and cultured for 24 hours and stained for HSV-1-GFP (green) and HSV-1 DNA (red) via RNAscope as described earlier. Representative images of merged HSV-1-GFP and HSV-1 DNA shown in three animals. The superficial epithelial cells are only well defined in Animal 4, under autofluorescence marking of the upper boundary. In Animals 2 and 3 microfolding obscures the upper layer of epithelial cells with flattened nuclei (C) Representative image of 20x and 100x magnification of patch-treated porcine corneal epithelium. Yellow arrows indicate HSV-1 DNA spreading along basal epithelium of the cornea with no penetration into the stroma. Scale bar = 50μm. (D) High magnification (100x) images of various stages of infection as indicated by HSV-1 DNA expression, including insets with orthogonal views. Insets show 1) diffuse cytoplasmic and 2) focal nuclear HSV-1 DNA staining. Arrows show focal cytoplasmic infection. Scale bar = 100 µm or as indicated. (E) High magnification image of individual/aggregate HSV-1 particles (white arrows) which may be exiting or between infected cells. Autofluorescent channel indicates epithelium is intact. Scale bar = 100 µm or as indicated. (F) Table summarizing the average number of HSV-1 DNA+ foci per mm of epithelium, the average width of each foci (µm) and the lateral spread as number of cells across the focus, (mean + range).

Fig 3. SPRR1A detected at the superficial and lower most epithelial layer of the porcine cornea.

Porcine corneas were subjected to mock conditions or HSV-1-treated (1 x 10⁸ PFU/mL) HD-MAPs and cultured for 24 hours. Tissue sections were labelled for HSV-1 DNA via RNAscope (red), SPRR1A (1:40) primary and donkey anti-rabbit AF647 secondary (1:400) (yellow) antibodies. Representative image of merged HSV-1 DNA and SPRR1A in two animals shown. Scale bar = 50μm.