<em>Ex Vivo</em> Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Abstract

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1-or HVEM-deficient mice. Video LinkThe video component of this article can be found at

Invasion of Herpes Simplex Virus Type 1 into Murine Epidermis: An Ex Vivo Infection Study

“…Mice, preparation of murine epidermis, isolation of primary cells, and viruses Epidermal sheets were taken from tail skin of adult wt (C57BL/6) or from ILK-K5 mice (Lorenz et al, 2007) as described previously (Petermann et al, 2015;Rahn et al, 2015). Briefly, after incubation of skin samples for 30 minutes at 37°C with 5 mg ml − 1 dispase II (Roche, Mannheim, Germany), the epidermis was removed from the dermis as an intact sheet.…”

“…Alternatively, skin samples were incubated for 4 hours at 37°C with 5 mM EDTA to remove the epidermis from the dermis. Epidermal sheets were incubated in DMEM/high glucose/ GlutaMAX (Life Technologies, Darmstadt, Germany) as described (Rahn et al, 2015). Infection was performed with purified preparations of HSV-1 wt strain 17 (Schelhaas et al, 2003).…”

“…In brief, virus inoculum was added to the cells at 37°C, defining time point 0. The calculation of the virus dose was based on the estimated cell number in the basal layer per epidermal sheet (Rahn et al, 2015).…”

“…To maintain murine melanocytes, primary cells were isolated from newborn epidermis as described (Rahn et al, 2015). The cell suspension was cultured on collagen-coated dishes (collagen G; Merck Millipore, Schwalbach, Germany) at 32°C and maintained in keratinocyte culture medium (DMEM/Ham's F-12 [3.5:1.1]; Merck Millipore) containing 50 μM calcium ions and 10% fetal calf serum (calcium-free), penicillin (400 IU ml − 1 ), streptomycin (50 μg ml − 1 ), adenine (1.8 × 10 4 M), glutamine (300 μg ml − 1 ), hydrocortisone (0.5 μg ml − 1 ), epidermal growth factor (10 ng ml − 1 ), cholera enterotoxin (10 10 M), insulin (5 μg ml − 1 ), and ascorbic acid (0.05 mg ml − 1 ) in the presence of mitomycin C-treated 3T3 fibroblasts (strain J2).…”

“…Upon infection with 10 PFU/cell, many hair germ cells became infected, whereas only a very limited number of infected cells was present in the bulge region that is the niche for the quiescent SCs (Figure 5a). Staining of the sebaceous glands was shown to be unspecific (Rahn et al, 2015). Raising the virus dose to 100 PFU/cell did not increase the number of infected cells in the bulge, suggesting that infection is indeed restricted to the hair germ cells (Figure 5a).…”