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

## TL;DR

A new pig cornea model shows that HSV-1 infection stays in the epithelium and does not spread deeper into the cornea, which could help improve treatments and vaccines for herpes keratitis.

## Contribution

A novel porcine corneal explant model demonstrates that HSV-1 infection is restricted to the epithelium and does not cross the anterior limiting membrane.

## Key findings

- HSV-1 infection and spread is confined to the corneal epithelium without reaching the stroma.
- SPRR1A expression in the uppermost epithelial layer and ALM suggests a barrier function against HSV-1.
- RNAscope detected HSV-1 infection stages in epithelial foci, confirming limited spread.

## 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.

Herpes simplex keratitis is a leading cause of blindness and a major indication for corneal transplantation. To examine the earliest stages of infection and spread of herpes simplex virus type 1 (HSV-1) we developed a model system in pig corneas in explant culture, using a highly sensitive method for detecting HSV DNA, RNAscope and fluorescently labelled virus. Corneas from pigs more closely resemble those from humans in size and structure than mice. HSV-1 infection required intraepithelial delivery by microneedle patch punctures and the virus was shown to spread throughout the corneal epithelium except for the outermost layer. HSV-1 also did not spread beyond the anterior limiting membrane (ALM) into the subjacent stroma. SPPR1A, which contributes to the barrier function of cornified epithelium of skin, was expressed by the uppermost epithelial layer and, in infected cornea, observed in the ALM, suggesting it contributes to limiting both superficial and deep HSV-1 spread. The various successive stages of nuclear and cytoplasmic HSV-1 infection were shown in foci of infection by RNAscope. Techniques used in this model can be selectively applied in scarce human corneas to verify key findings and ultimately improve therapy for herpes keratitis and vaccine design to prevent it.