Researchers at the University of Iowa (UI) Health Care and colleagues at the Texas Biomedical Research Institute, and Boston University, used human skin explants to track the cellular route that the deadly Ebola virus (EBOV) uses to traverse the inner and outer layers of skin and emerge onto the skin’s surface during the final stages of infection. The study found that the virus infects at least three cell types in the lower dermis and then permeates the outermost epidermis via infiltration of another cell type. As well as demonstrating that human skin specimens actively support EBOV infection, the study results also suggest that the skin’s surface may be one route of person-to-person transmission. The team also noted the potential utility of their skin model system for antiviral studies, and as a platform for testing drugs.
“The skin is the largest organ in the human body yet is woefully understudied compared to most other organs,” said Wendy Maury, PhD, UI professor of microbiology and immunology. “Interactions of EBOV with skin cells have not previously been extensively examined … Our work provides evidence for one mechanistic avenue that EBOV uses to exit from the human body. A comprehensive understanding of which cells are targeted during virus infection is critical for rational development of antiviral approaches.”
Maury is senior author of the team’s published paper in Science Advances, titled “Multiple cell types support productive infection and dynamic translocation of infectious Ebola virus to the surface of human skin,” in which they stated “Our findings define a route through which infectious virus traverses the skin to the epidermal surface, thereby potentially contributing to person-to-person transmission.”
Ebola is hemorrhagic disease caused by the Ebola virus, which is endemic in parts of East-Central and West Africa. “The characteristic signs of EBOV include hemorrhagic fever, gastrointestinal symptoms, and multiple organ dysfunction syndrome,” the authors wrote. A primary route for person-to-person transmission is through contact with bodily fluids from an infected person, the team further explained. “EBOV is spread by direct contact with an infected individual or their body fluids, with mucosal transmission considered to be an important route.”
Recent Ebola outbreaks, including the 2013–2016 Ebola epidemic in West Africa, in addition demonstrated that infectious Ebola virus is also found on the skin’s surface of those who have succumbed to infection or at late times during infection. Past studies in animal models have corroborated these observations. “It has also been established that EBOV RNA and infectious virus are on the skin surface at later stages of infection and EBOV antigen and/or RNA is present in skin samples from humans and experimentally infected animals,” the investigators pointed out. And while evidence suggests that EBOV can be passed on from skin contact with a person in the later stages of the disease, very little is known about how the virus makes its way out of the body and onto the skin’s surface. “… the mechanism(s) by which the virus travels to the skin surface, and the skin cells responsible for supporting infection, are not fully understood,” the investigators further noted. “The lack of robust and reproducible models to study EBOV skin infection is a major barrier to defining the role of the skin in viral load and transmission.”
To overcome this barrier the team, led by Maury and Kelly Messingham, PhD, UI research professor of dermatology, developed a new approach to examine which cells within the skin are infected by Ebola virus. They created a human skin explant system using full-thickness skin biopsies from healthy individuals, which contained both deeper (dermal), and surface (epidermal) layers of skin.
To study how Ebola virus moves through skin, the explants were placed dermal side down in culture media and virus particles were added to the media so that they entered the skin from the underside, modeling virus egress from the blood to the surface of the skin. The researchers used virus-tracing and cell-tagging techniques to follow the journey of the virus through the skin layers to the upper surface of the skin, identifying which cells were infected over time.
They examined the ex vivo human tissue viral cultures over the course of 17 days, noting how the virus traveled by infecting select cell groups from the undermost dermis to the outermost, or apical, epidermis. Endothelial, myeloid, and fibroblast cells in the dermis were primarily positive for Ebola antigen. Within three days, the virus reached the apical epidermis and populated cells there called keratinocytes. “In the dermis, cells of myeloid, endothelial, and fibroblast origin were EBOV antigen–positive whereas keratinocytes harbored virus in the epidermis,” the investigators wrote. Detection of infectious virus on the epidermal surface within three days indicated that the virus rapidly spreads and moves through the explants to the skin’s surface.
While some of the cell types identified are also found to be infected by EBOV in other organs, keratinocytes, which are unique to the skin, had not been previously appreciated to support EBOV infection. Interestingly, the study found that virus replication was more robust in the epidermal layer than the dermal layers on a per gram basis. “We identify permissive dermal and epidermal cell subsets that support EBOV infection and demonstrate that EBOV spreads through the skin from the basal surface of the explant, trafficking through the dermis and epidermis to the apical surface of the skin,” the authors stated. “These findings may explain person-to-person transmission via skin contact.”
The team performed additional tests using purified human keratinocytes and fibroblasts to see which intracellular mechanisms supported Ebola infiltration. They found that the endosomal protein, NPC1, and the phosphatidylserine receptor, AXL, were critical. “Purified human fibroblasts and keratinocytes supported EBOV infection ex vivo and both cell types required the phosphatidylserine receptor, AXL, and the endosomal protein, NPC1, for virus entry.”
Messingham concluded, “This study explores the role of the skin as a potential route of Ebola virus infection and identifies, for the first time, several cell types in the skin that are permissive to infection. In total, these findings elucidate a mechanism by which EBOV traffics to the skin’s surface and may explain person-to-person transmission via skin contact.”
The study in addition demonstrated that human skin explants can serve as complex, three- dimensional organ models for studying the efficacy of antivirals against EBOV, offering up a new, highly useful, and inexpensive model system for therapeutic testing. “These explants serve as excellent tissue models for examining potential antivirals,” the scientists wrote. “We propose that this accessible and relevant tissue model should be incorporated into antiviral workflows as a highly useful step to evaluate drug efficacy before animal studies.”
