# Herpes simplex virus assembly and spread in murine skin after infection from the outside

## TL;DR

A new mouse skin model shows how herpes simplex virus spreads in the skin without damaging tissue, offering insights into early infection and treatment.

## Contribution

A novel ex vivo model for HSV-1 infection in murine skin that avoids tissue disruption and enables study of early viral spread.

## Key findings

- HSV-1 spreads exclusively in the epidermis, forming expanding infection centers with hundreds of infected keratinocytes.
- Advanced microscopy revealed intracellular assembly of HSV-1 structures, including nuclear capsids and enveloped virions.
- The model maintains skin integrity and allows tracking of viral spread and mutant phenotypes in a physiological context.

## Abstract

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation without further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently tagged capsid protein VP26, and observed the formation of nuclear capsid assembly sites and nuclear capsid egress and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By using electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model.

This study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers increasing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.