De La Cruz Nydia C, Möckel Maureen, Wirtz Lisa, Knebel-Mörsdorf Dagmar
Center for Biochemistry, University Hospital Cologne, University of Cologne, Cologne, Germany.
Department of Pediatrics, University Hospital Cologne, University of Cologne, Cologne, Germany.
Bio Protoc. 2022 May 5;12(9):e4411. doi: 10.21769/BioProtoc.4411.
Although herpes simplex virus 1 (HSV-1) is a well-studied virus, how the virus invades its human host via skin and mucosa to reach its receptors and initiate infection remains an open question. For studies of HSV-1 infection in skin, mice have been used as animal models. Murine skin infection can be induced after injection or scratching of the skin, which provides insights into disease pathogenesis but is clearly distinct from the natural entry route in human tissue. To explore the invasion route of HSV-1 on the tissue level, we established an infection assay using skin explants. Here, we detail a protocol allowing the investigation of how the virus overcomes mechanical barriers in human skin to penetrate in keratinocytes and dermal fibroblasts. The protocol includes the preparation of total skin samples, skin shaves, and of separated epidermis and dermis, which is followed by incubation in virus suspension. The infection assay allows the visualization, quantification, and characterization of single infected cells in the epidermis and dermis prior to viral replication and the virus-induced tissue damage. Hence, this experimental approach enables the identification of primary viral entry portals. Graphical abstract.
尽管单纯疱疹病毒1型（HSV-1）是一种已被充分研究的病毒，但该病毒如何通过皮肤和黏膜侵入人类宿主以抵达其受体并引发感染仍是一个悬而未决的问题。在研究HSV-1在皮肤中的感染时，小鼠被用作动物模型。通过注射或抓挠皮肤可诱导小鼠皮肤感染，这为疾病发病机制提供了见解，但显然与病毒在人体组织中的自然进入途径不同。为了在组织水平上探索HSV-1的侵入途径，我们建立了一种使用皮肤外植体的感染试验。在此，我们详细介绍一种方案，该方案可用于研究病毒如何克服人类皮肤中的机械屏障以侵入角质形成细胞和真皮成纤维细胞。该方案包括制备全皮样本、剃除的皮肤以及分离的表皮和真皮，随后将其置于病毒悬液中孵育。该感染试验能够在病毒复制和病毒诱导的组织损伤之前，对表皮和真皮中单个感染细胞进行可视化、定量和表征。因此，这种实验方法能够识别病毒的主要进入门户。图形摘要。
