Byung-Joon Seung ¹ Sushil Khatiwada ¹ Daniel L Rock ¹ Gustavo Delhon ^2*

• ¹ University of Illinois at Urbana-Champaign, Champaign, United States
• ² University of Nebraska-Lincoln, Lincoln, Nebraska, United States

Reflecting their tropism for keratinocytes, most poxviruses infecting vertebrates infect, replicate to high titers, and cause pathology in the skin. Keratinocytes, the main cells of the epidermis, are found in different stages of a differentiation program that produces the critical barrier against environmental damage. While systemic poxviruses (e.g. smallpox virus, sheeppox virus) also infect other cell types, the parapoxvirus orf virus (ORFV), which causes localized infections in sheep and goats, has not been shown to replicate in cells other than keratinocytes. Notably, ORFV infection only occurs after-or concomitant with epidermal damage and subsequent healing response and shows unexplained delayed virus replication in an uncharacterized keratinocyte subpopulation. Using in situ hybridization, immunohistochemistry, confocal microscopy, qPCR, and a full-thickness wound/infection model in sheep, the natural host, we show that during an initial two-day eclipse phase viral transcription and viral DNA replication are not detected. Between day 2 and 3 pi, viral transcription is first detected in keratinocytes of the stratum granulosum and upper stratum spinosum at the proliferative zone in the wound margin. These cells are positive for cytokeratin 10, a suprabasal marker; cytokeratin 6, a protein induced during early repair responses; stratum granulosum markers filaggrin and loricrin; and negative for the nuclear proliferation marker Ki-67 and cytokeratin 14, a basal cell marker. This marker profile suggests that keratinocytes supportive of viral replication are engaged in advanced keratinocyte differentiation rather than proliferation.