Infection with herpes simplex virus type 1 (HSV-1) causes the most common skin disease. Various test systems have been established to recapitulate this cyclical pathway of productive infection, latency, and reactivation. Most studies of latency and reactivation are conducted in animal models. However, the small number of neurons which harbor the viral genome, the complexity of the in vivo setting, and ethical constraints place limits on animal studies. So far, no in vitro model which resembles natural latency exists. Here, we describe the first in vitro HSV-1 infection model based on a human skin equivalent. The 3D infection model is generated using the human keratinocyte cell line HaCaT grown on a collagen substrate containing human primary fibroblasts and in addition a quiescently HSV-1 infected neuronal component.
Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm2.
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