title: Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture.
abstract: This study describes the outcome of herpes simplex virus type 1 (HSV-1) infection in an organotypic raft culture of spontaneously immortalized HaCat keratinocytes and human fibroblasts, as related to the virus load and epithelial stratification and differentiation. In this model, a confluent monolayer of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV-1 before induction of differentiation by lifting of the culture to the air-liquid interface always resulted in a productive infection, but the virus yield was highest when the inoculation took place 72 h after seeding. Even at 0.1 p.f.u. per culture, the HaCat cultures became HSV positive. Infection of the full-thickness epithelium at 5 p.f.u. per culture resulted in a productive infection of the whole epithelium. The HaCat cells were about 10 times more sensitive to HSV-1 infection than the Vero cells in which the virus stocks were titrated. The raft cultures infected 30 min after lifting were negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocytochemistry. PCR showed the presence of HSV-1 DNA and in situ hybridization showed reactivity with a latency-associated RNA probe, indicating the presence of a non-productive infection. Two different patterns of virus spread in epithelia were found: (i) lateral spread through the superficial layers of the epithelium and (ii) a demarcated infection throughout the whole thickness of the epithelium at the margins of the culture.
authors.affiliation: Department of Virology1, MediCity Research Laboratory2 and Department of Oral Pathology, Institute of Dentistry3, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland.
authors.forename: Veijo
authors.initials: V
authors.lastname: Hukkanen
authors.affiliation: Department of Virology1, MediCity Research Laboratory2 and Department of Oral Pathology, Institute of Dentistry3, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland.
authors.forename: Hannamari
authors.initials: H
authors.lastname: Mikola
authors.affiliation: Department of Virology1, MediCity Research Laboratory2 and Department of Oral Pathology, Institute of Dentistry3, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland.
authors.forename: Marja
authors.initials: M
authors.lastname: Nykänen
authors.affiliation: Department of Virology1, MediCity Research Laboratory2 and Department of Oral Pathology, Institute of Dentistry3, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland.
authors.forename: Stina
authors.initials: S
authors.lastname: Syrjänen
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004847:Epithelial Cells / Q000821:virology; D005347:Fibroblasts / Q000166:cytology / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000821:virology*; D013997:Time Factors; D014709:Vero Cells; D019562:Viral Load; D014779:Virus Replication