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Title: INHIBITION OF PROTEASE-RESISTANT PRION PROTEIN FORMATION IN A TRANSFORMED DEER CELL LINE INFECTED WITH CHRONIC WASTING DISEASE

Author
item RAYMOND, GREGORY - NIAID, NIH, HAMILTON, MT
item OLSEN, EMILY - NIAID, NIH, HAMILTON, MT
item LEE, KIL SUN - NIAID, NIH, HAMILTON, MT
item RAYMOND, LYNNE - NIAID, NIH, HAMILTON, MT
item Bryant, Paul
item BARON, GERALD - NIAID, NIH, HAMILTON, MT
item CAUGHEY, WINSLOW - NIAID, NIH, HAMILTON, MT
item KOCISKO, DAVID - NIAID, NIH, HAMILTON, MT
item McHolland, Linda
item FAVARA, CYNTHIA - NIAID, NIH, HAMILTON, MT
item LANGEVELD, JAN P.M. - LELYSTAD, THE NETHERLANDS
item VAN ZIJDERVELD, FRED - LELYSTAD, THE NETHERLANDS
item Mayer, Richard
item MILLER, MICHAEL - COLO DIVISION OF WILDLIFE
item WILLIAMS, ELIZABETH - UW DEPT OF VET SCI
item CAUGHEY, BYRON - NIAIN, NIH, HAMILTON, MT

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/17/2005
Publication Date: 1/1/2006
Citation: Raymond, G.J., Olsen, E.A., Lee, K., Raymond, L.D., Bryant III, P.K., Baron, G.S., Caughey, W.S., Kocisko, D.A., Mcholland, L.E., Favara, C., Langeveld, J., Van Zijderveld, F.G., Mayer, R.T., Miller, M.W., Williams, E.S., Caughey, B. 2006. Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease. Journal of Virology. 80(2):1-9.

Interpretive Summary: Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Technical Abstract: Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.