A rapid accurate culture assay for infectivity in Transmissible Encephalopathies.
The molecular and structural features of infectious agents that cause CJD, scrapie and BSE remain controversial. A major impediment for agent resolution is the very long and expensive animal assays of infectivity. It is crucial to develop a rapid and broadly applicable cell culture assay to titer and compare different TSE agent strains. Because we found GT1 hypothalamic cells, unlike neuroblastoma N2a clones, were highly susceptible to a variety of TSE agents, and could stably produce high agent titers for >1 year, we studied the progressive display of abnormal prion protein (PrP-res) in GT1 cells following exposure to serially diluted 22L scrapie brain homogenates; PrP-res was used as a surrogate, but non-quantitative marker of GT1 infection. Even as early as the first cell split after 22L exposure, GT1 cells produced their own PrP-res bands that were clearly different than brain bands. Plots from passages 3-7 showed a good discrimination of 3 fold differences in titer over a range of >2 logs, with the same endpoint sensitivity (2 x 10(8) LD(50)/gm) as animal assays. Interestingly, the rapid production of de novo PrP-res suggested that GT1 PrP-res might be induced by interaction with an early-intermediate form of a particle that was not fully infectious. The GT1 assay here was also invaluable for rapidly identifying cell cultures with variant titers, even after detergent lysis. Additionally, in-situ PrP amyloid staining provided an independent measure of the minimum infectious dose per cell. Standardized GT1 assays can be used for direct comparison of different agent strains, and will facilitate the rapid isolation of essential agent components.
Liu
Ying
Y
Section of Neuropathology, Yale Medical School, New Haven, CT 06510, USA.
Sun
Ru
R
Chakrabarty
Trisha
T
Manuelidis
Laura
L
D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods*; D051379:Mice; D018620:PrPSc Proteins / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism / Q000472:pathogenicity*; D017096:Prion Diseases / Q000378:metabolism* / Q000473:pathology; D011328:Prions / Q000378:metabolism; D012608:Scrapie / Q000378:metabolism; D012756:Sheep
D018620:PrPSc Proteins; D011328:Prions