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Title:   Author  item
                                            MUNOZ-GUTIERREZ, JUAN - Washington State Universityitem
                                            Schneider, Daviditem
                                            BASZLER, T - Washington State Universityitem
                                            DINKEL, K - Washington State Universityitem
                                            Greenlee, Justinitem
                                            Nicholson, Ericitem
                                            STANTON, JAMES - Washington State University
  Submitted to: Virus Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 10/20/2014
                                    Publication  Date: 1/12/2015
                                    Citation: Munoz-Gutierrez, J.F., Schneider, D.A., Baszler, T.V., Dinkel, K.D., Greenlee, J.J., Nicholson, E.M., Stanton, J.J. 2015. hTERT-immortalized ovine microglia propagate natural scrapie isolates. Virus Research. 198:35-43.Interpretive Summary: Scrapie is a transmissible, fatal brain disease of sheep that is caused by a novel infectious agent called a prion. While rodent-derived cell culture systems have aided prion research, these systems remain poorly sensitive and variable to infection by natural prion isolates, for example, those found in the brain of sheep with scrapie. In an attempt to overcome this limitation, we established cultures of sheep brain-derived microglia, a cell type naturally infected during the course of disease. The limited lifespan of such primary cell lines, however, necessitates periodic isolation and characterization of new cells from additional animals. In this study, we “immortalized” cultured sheep microglia by introducing into these cells a new gene, telomerase reverse transcriptase. In addition, we identified immortalized microglia that were sensitive to infection by scrapie prions in the brain of several naturally-infected sheep originating from different regions of the United States. These findings represent a major improvement in the efficiency of maintaining cells in culture that retain sensitivity to infection with natural prion isolates. As compared to rodent-derived systems, we anticipate improved translation of studies utilizing these sheep brain cells to scrapie disease in the natural host.
                                Technical Abstract: Scrapie is a transmissible fatal neurodegenerative disease of sheep that is caused by a novel infectious agent called a prion. The study of prions under controlled culture conditions is crucial to advancing knowledge regarding prion processes of replication and accumulation within cells. Unfortunately, several well-established rodent cell culture systems remain poorly sensitive to infection by natural sources of prions, including brain material from sheep with scrapie, and some research conducted using these models have failed to directly translate to disease in animals. In this study, a primary cell line of ovine brain-derived microglia were immortalized by transfection with human telomerase reverse transcriptase (hTERT) gene. Functional expression of hTERT was confirmed and multiple sublines of hTERT-microglia have since been passaged multiple times beyond the culture lifespan of non-transfected parental cells. Several sublines of hTERT-microglia were permissive to culture-adapted scrapie prions, and a comparison with non-permissive sublines revealed a correlation of permissiveness with the quality (truncated isoforms) rather than the total quantity of expressed cellular prion protein (PrP-C). Further, one subline was shown to be permissive to several natural prion isolates from sheep with scrapie disease. In comparison to rodent-derived systems, we anticipate that results obtained using ovine hTERT-microglia and natural isolates of scrapie prions will better translate to prion diseases in animals.


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