title: hTERT-immortalized ovine microglia propagate natural scrapie isolates.
abstract: Ex vivo propagation of natural prion isolates (i.e., propagated solely in the natural host) is crucial for the characterization and study of transmissible spongiform encephalopathies (TSEs). Several well-established, prion-permissive cell culture systems are available; however, only a few cell lines are permissive to natural prion isolates and these cells are not pathophysiologically relevant (e.g., renal epithelium and fibroblast-like cells). Therefore, a pathophysiologically relevant cell line derived from a natural TSE host could be used for propagation of natural prion isolates. In this study, ovine brain macrophages (microglia) were immortalized by transfection with the human telomerase reverse transcriptase (hTERT) gene to identify cell lines (hTERT-microglia) permissive to natural scrapie prion isolates. Following transfection, hTERT-microglia were passaged up to 100 times and their lifespan was significantly longer compared to parental cells (Fisher's exact test, P<0.001). Multiple sublines were permissive to cell culture-adapted prions; two sublines were also permissive to natural scrapie isolates (i.e., derived from brain homogenates of sheep infected with scrapie). Prion infectivity and partial protease resistance of the prion protein were maintained in hTERT-microglia. Comparisons between scrapie-permissive and non-permissive hTERT-microglia sublines revealed that overall quantity of the normal cellular prion protein was not associated with prion permissiveness. The use of hTERT-microglia in future TSE studies may be more germane to the characterization of the cellular and subcellular pathophysiology of natural scrapie prion isolates and to investigate host-specific factors involved in prion replication.
authors.affiliation: Department of Microbiology and Pathology, College of Veterinary Medicine, Washington State University, PO Box 64700, Pullman, WA 99164-7010, United States. Electronic address: jmunoz@vetmed.wsu.edu.
authors.forename: Juan F
authors.initials: JF
authors.lastname: Muñoz-Gutiérrez
authors.affiliation: Department of Microbiology and Pathology, College of Veterinary Medicine, Washington State University, PO Box 64700, Pullman, WA 99164-7010, United States; United States Department of Agriculture, Agricultural Research Service, Pullman, WA 99164-6630, United States. Electronic address: das@vetmed.wsu.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Schneider
authors.affiliation: Department of Microbiology and Pathology, College of Veterinary Medicine, Washington State University, PO Box 64700, Pullman, WA 99164-7010, United States. Electronic address: baszlert@vetmed.wsu.edu.
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Baszler
authors.affiliation: National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, PO Box 701920, Dayton Ave., Ames, IA, United States. Electronic address: justin.greenlee@ars.usda.gov.
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Greenlee
authors.affiliation: National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, PO Box 701920, Dayton Ave., Ames, IA, United States. Electronic address: eric.nicholson@ars.usda.gov.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Nicholson
authors.affiliation: Department of Microbiology and Pathology, College of Veterinary Medicine, Washington State University, PO Box 64700, Pullman, WA 99164-7010, United States. Electronic address: jbs@uga.edu.
authors.forename: James B
authors.initials: JB
authors.lastname: Stanton
mesh_terms: D000818:Animals; D002460:Cell Line; D005260:Female; D008297:Male; D017628:Microglia / Q000201:enzymology*; D012608:Scrapie / Q000378:metabolism*; D012756:Sheep
keywords: Immortalization; Microglia; Prion; Scrapie; hTERT