Progress 09/12/07 to 09/11/12
Outputs
Progress Report Objectives (from AD-416): To develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate mechanisms of prion conversion and develop new diagnostic methods for prion diseases. Approach (from AD-416): Uninfected stable ovine and cervid cell lines, from genetically susceptible animals, will be developed and then assayed for the ability to accumulate PrPd after inoculation with the TSE agent. Cells will originate from tissues that accumulate PrPd in natural disease, namely reproductive tissue (placental trophoblasts) and brain (microglia and astrocytes). Preliminary work shows successful immortalization and culture of ovine microglia and demonstration of prion accumulation in the cell cultures following exposure to scrapie agent. The PrPd accumulating cells lines will be used for comparative transcriptomic experiments toward identifying accessory molecules for prion conversion and mechanisms of genetic resistance to prion conversion. Unique gene expression profiles in PrPd-accumulating cells will be identified using bovine gene chip expression arrays and specific prion conversion associated genes identified by RNA interference. The findings of the proposed study have applications to both human and veterinary medicine and will increase understanding of the pathogenesis of prion diseases by defining the genetic response to prion accumulation. Identification of cofactors involved in the accumulation of PrPSc will identify putative targets for therapeutic intervention and potential biomarkers of infection. Finally, the development of PrPSc-accumulating sheep cell lines could provide an in vitro infectivity assay for prion disease diagnosis and a model to further study pathogenesis using cells from a natural host species. This work relates to sub-objective 1.1 of parent project in proving data supporting improved control and eradication efforts. This is the final report for this SCA which expires in September 2012, which was to develop and optimize novel cell lines for in vitro amplification of abnormal prion protein in order to investigate the mechanisms of prion conversion and develop new diagnostic methods for prion disease. Two primary cell lines of differing tissue origins were isolated from sheep, including sheep with different prion protein genotypes. The cell lines were characterized and tested for permissiveness to authentic sources of scrapie prions, with comparison to a widely utilized ovinized transgenic rabbit cell line. The effects of co-viral infection with either small ruminant lentivirus or pestivirus on primary ovine microglial cell propagation and accumulation of abnormal prion proteins were investigated. Further, the novel anti-prion effects of a series of compounds having anti-pestivirus effects were investigated. In a transcriptome study of the primary ovine microglial cell line, it was determined that the expression levels of thirty-nine genes were altered during prion propagation and accumulation. To overcome significant limitations of using primary cell lines, the ovine microglial cell line was immortalized, re-characterized and tested for permissiveness to authentic sources of scrapie prions and small ruminant lentivirus and pestivirus. The project was monitored by the lead scientist through frequent meetings with the scientific staff to review recent progress and coordinate research activities.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) To develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate mechanisms of prion conversion and develop new diagnostic methods for prion diseases. Approach (from AD-416) Uninfected stable ovine and cervid cell lines, from genetically susceptible animals, will be developed and then assayed for the ability to accumulate PrPd after inoculation with the TSE agent. Cells will originate from tissues that accumulate PrPd in natural disease, namely reproductive tissue (placental trophoblasts) and brain (microglia and astrocytes). Preliminary work shows successful immortalization and culture of ovine microglia and demonstration of prion accumulation in the cell cultures following exposure to scrapie agent. The PrPd accumulating cells lines will be used for comparative transcriptomic experiments toward identifying accessory molecules for prion conversion and mechanisms of genetic resistance to prion conversion. Unique gene expression profiles in PrPd-accumulating cells will be identified using bovine gene chip expression arrays and specific prion conversion associated genes identified by RNA interference. The findings of the proposed study have applications to both human and veterinary medicine and will increase understanding of the pathogenesis of prion diseases by defining the genetic response to prion accumulation. Identification of cofactors involved in the accumulation of PrPSc will identify putative targets for therapeutic intervention and potential biomarkers of infection. Finally, the development of PrPSc-accumulating sheep cell lines could provide an in vitro infectivity assay for prion disease diagnosis and a model to further study pathogenesis using cells from a natural host species. During FY2011, collaborative work between ADRU scientists and faculty at Washington State University was focused on the mechanisms of enhanced prion conversion in cell culture systems during viral co-infection. The work to date includes comparison of different types of viruses on prion conversion as well as testing effects novel anti-viral and anti-prion drugs. The project is monitored by the lead scientist through frequent meetings with the scientific staff to review recent progress and coordinate research activities.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) To develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate mechanisms of prion conversion and develop new diagnostic methods for prion diseases. Approach (from AD-416) Uninfected stable ovine and cervid cell lines, from genetically susceptible animals, will be developed and then assayed for the ability to accumulate PrPd after inoculation with the TSE agent. Cells will originate from tissues that accumulate PrPd in natural disease, namely reproductive tissue (placental trophoblasts) and brain (microglia and astrocytes). Preliminary work shows successful immortalization and culture of ovine microglia and demonstration of prion accumulation in the cell cultures following exposure to scrapie agent. The PrPd accumulating cells lines will be used for comparative transcriptomic experiments toward identifying accessory molecules for prion conversion and mechanisms of genetic resistance to prion conversion. Unique gene expression profiles in PrPd-accumulating cells will be identified using bovine gene chip expression arrays and specific prion conversion associated genes identified by RNA interference. The findings of the proposed study have applications to both human and veterinary medicine and will increase understanding of the pathogenesis of prion diseases by defining the genetic response to prion accumulation. Identification of cofactors involved in the accumulation of PrPSc will identify putative targets for therapeutic intervention and potential biomarkers of infection. Finally, the development of PrPSc-accumulating sheep cell lines could provide an in vitro infectivity assay for prion disease diagnosis and a model to further study pathogenesis using cells from a natural host species. Documents SCA with WSU. Significant Activities that Support Special Target Populations The purpose of this new SCA is to develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate the mechanisms of prion conversion and develop new diagnostic methods for prion disease. A change in the transcriptional profile of cultured ovine microglia near maximal PrPSc accumulation has now been quantified using the Affymetrix Bovine Genome Array. Thirty-nine genes with altered expression were identified. The project is monitored by the ADODR by frequent meetings with the scientific staff to review recent progress and coordinate research activities.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) To develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate mechanisms of prion conversion and develop new diagnostic methods for prion diseases. Approach (from AD-416) Uninfected stable ovine and cervid cell lines, from genetically susceptible animals, will be developed and then assayed for the ability to accumulate PrPd after inoculation with the TSE agent. Cells will originate from tissues that accumulate PrPd in natural disease, namely reproductive tissue (placental trophoblasts) and brain (microglia and astrocytes). Preliminary work shows successful immortalization and culture of ovine microglia and demonstration of prion accumulation in the cell cultures following exposure to scrapie agent. The PrPd accumulating cells lines will be used for comparative transcriptomic experiments toward identifying accessory molecules for prion conversion and mechanisms of genetic resistance to prion conversion. Unique gene expression profiles in PrPd-accumulating cells will be identified using bovine gene chip expression arrays and specific prion conversion associated genes identified by RNA interference. The findings of the proposed study have applications to both human and veterinary medicine and will increase understanding of the pathogenesis of prion diseases by defining the genetic response to prion accumulation. Identification of cofactors involved in the accumulation of PrPSc will identify putative targets for therapeutic intervention and potential biomarkers of infection. Finally, the development of PrPSc-accumulating sheep cell lines could provide an in vitro infectivity assay for prion disease diagnosis and a model to further study pathogenesis using cells from a natural host species. Documents SCA with WSU. Significant Activities that Support Special Target Populations Development of Sensitive In vitro Techniques for Prion Detection: Project Number 5348-32000-026-08S. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Washington. Additional details of research can be found in the report for the parent project 5348-32000-026-00D Transmissible Spongiform Encephalopathies: the role of genetics, strain variation, and environmental contamination in disease control. The purpose of this new SCA is to develop ovine and cervid cell lines for in vitro amplification of abnormal prion protein in order to investigate the mechanisms of prion conversion and develop new diagnostic methods for prion disease. Primary cell lines of ovine microglial cells were shown to propagate and accumulate abnormal prion proteins. Prion protein levels were higher in cell lines co-infected with a small ruminant lentivirus. The project is monitored by the ADODR by frequent meetings with the scientific staff to review recent progress and coordinate research activities.
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