Background

Herpes simplex virus 1 (HSV-1) is one of the most prevalent human pathogens. During primary infection, the virus invades skin and mucocutaneous sites, which is followed by the latent infection of sensory neurons. Upon reactivation, HSV-1 travels back to near the initial infection site and can cause lesions. As the skin and mucosa form a protective barrier to infection, the general assumption is that the virus infects its host organism via epithelial breaks due to mechanical injuries or pathophysiological modifications. Viral replication largely takes place in the epithelium, and the extent of productive infection is restricted by the immune response. How HSV-1 enters single cells in culture is extensively studied; however, we know less about the viral invasion in human skin and mucosa to initiate infection in skin cells. Thus, the conditions under which the virus overcomes the epithelial barriers, either during primary or recurrent infection, and gains access to its receptors on the skin cells in vivo, are yet to be elucidated.

To address HSV-1 infection in tissue, initial studies were performed in 3-dimensional (3D) skin cultures giving first insights into how the virus may spread and how efficiently differentiating human keratinocytes can be infected (Syrjänen et al., 1997; Visalli et al., 1997; Hukkanen et al., 1999). More recently, human mucosa and skin explants have been used as ex vivo HSV-1 infection models, which allow studying various steps of viral infection much closer to the in vivo situation than 3D cultures or animal models. To analyze the determinants of viral invasion, we ex vivo infected human oral mucosa and detected no infected cells in complete mucosa samples but only invasion in the basal epithelial layer once the connective tissue and the basement membrane were removed (Thier et al., 2017). Intriguingly, mechanical wounding of the mucosa samples is insufficient for HSV-1 to overcome the epithelial barriers (Thier et al., 2017). However, successful HSV-1 infection was observed in human abdominal skin after ex vivo infection of microneedle-pretreated skin, thus providing a model to study the efficiency of antiviral drugs (Tajpara et al., 2018). Tajpara and colleagues (2018) infected thin dermatome-cut skin with lesions partly penetrating both epidermis and dermis and showed the pathogenesis of HSV-1 4 days after infection. The assumption is that viral invasion occurs via the sample edges and at those lesions that cross the skin sample, which, in turn, leads to extensive tissue damage over time. To analyze the initial sites of HSV1 invasion in human skin upon wounding and address the susceptibility of the epidermal layers and the dermis, we developed the protocol described here (De La Cruz et al., 2021). The procedure is based on our ex vivo infection studies in murine skin and human oral mucosa (Rahn et al., 2015a, 2015b; Thier et al., 2017; Wirtz et al., 2020; De La Cruz and Knebel-Mörsdorf, 2021). Our focus is on the early events of infection to understand how the virus overcomes the distinct epidermal barriers, identify which cells are initially infected, and determine how the virus spreads in tissue. As murine and human total skin samples are protected against ex vivo infection from the apical site confirming the protective barrier of healthy skin (Rahn et al., 2015b; De La Cruz et al., 2021), we dissected the skin samples to investigate how susceptible the epidermis and dermis are. After separation of the dermis from the epidermis by dispase II treatment, the basement membrane is disrupted, and remaining components are only occasionally found on the apical site of the dermis and the basal layer of the epidermis. Infection of the separated human epidermis with high virus dose leads to infection of basal and suprabasal keratinocytes, which results in cytopathic effects at 24 h postinfection (p.i.) (De La Cruz et al., 2021). The onset of viral infection in single basal keratinocytes and viral spreading in suprabasal layers can be visualized by immunostaining of very early expressed viral genes such as ICP0 and ICP4. Alternatively, virus particles could be stained or tagged HSV-1 strains could be used for infection. The challenge is to visualize single particles in tissue prior to viral gene expression, to differentiate between noninfectious and infectious particles, and to localize particles in- or outside of cells. As the human epidermis is highly susceptible to HSV 1, virus titers can be determined at various times p.i. In contrast to the epidermis, the infection protocol results only in single infected fibroblasts in the human dermis even at 24 h p.i. (De La Cruz et al., 2021).

In addition, the skin samples could be mechanically wounded or stimulated with cytokines prior to ex vivo infection. Further modifications of the skin surface could be achieved by pre-incubation with bacterial or viral pathogens.

Our infection protocol is most suitable for investigating the early steps of HSV-1 infection in tissue. These include the interaction with epidermal barriers and with components of the extracellular matrix, the role of cellular receptors that allow viral penetration, and the cellular response to initial infection. The dissection of the human skin in skin shaves, epidermis, and dermis offers various experimental tools to address questions from different perspectives.