Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1.
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.
Center for Biochemistry, University of Cologne.
Elena
E
Rahn
Center for Biochemistry, University of Cologne.
Katharina
K
Thier
Center for Biochemistry, University of Cologne.
Philipp
P
Petermann
Center for Biochemistry, University of Cologne; Department of Dermatology, University of Cologne; dagmar.moersdorf@uni-koeln.de.
Dagmar
D
Knebel-Mörsdorf
D000818:Animals; D004817:Epidermis / Q000821:virology*; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000472:pathogenicity*; D051379:Mice; D017193:Skin Diseases, Viral / Q000821:virology*