SUMMARYA rat phaeochromocytoma cell line, termed PC12, was used to study scrapie replication. These cells, in response to the addition of nerve growth factor (NGF), exhibit a number of neuronal properties including morphological differentiation, electrophysiological responsiveness, and neurotransmitter synthesis. Cultures were exposed to scrapie brain homogenate (strain 139A), harvested every week for up to 6 weeks, and assayed for scrapie infectivity. Scrapie replication in vitro was monitored by injecting scrapie agent-exposed NGF-treated PC 12 cells into mice and measuring time intervals from injection to onset of clinical symptoms. Mouse incubation periods vary inversely with the amount of scrapie infectivity present. Cells harvested at 7 and 14 days after exposure to scrapie agent showed a decrease in the level of infectivity followed by an increase at subsequent time points. The increase in scrapie infectivity from early to late time intervals after agent exposure clearly indicated replication in vitro. A fusion agent was not necessary to establish infection, and the addition of mouse peritoneal macrophages caused a reduction in the yield of infectivity per culture. Examination of cells by phase-contrast microscopy failed to reveal any cytopathology.

Several inbred strains of mice were injected with different scrapie agents and their total body weight was monitored throughout the incubation period. As a control, mice were injected with normal mouse brain homogenate. For most combinations of scrapie agent and mouse strain, weights during the preclinical phase were similar to or lower than the average weight of controls. For some combinations there was a significant increase in weight (compared to controls) during the latter part of the preclinical phase of disease. The effect was dependent on both agent and mouse strain, i. e., in some cases a mouse strain showed the increase with one scrapie agent but not another and some scrapie agents caused the increase in one inbred strain of mouse but not in another strain. The increase in weight was due to accumulations of fat rather than a generalized increase in weight of various organs. With one mouse strain (SJL), there was increased vacuolation seen in the hypothalamus of mice injected with scrapie agents that showed the increase in weight compared to the lesion intensity with an agent which did not cause the weight increase.

Mouse peritoneal macrophages were exposed to scrapie (ME7 strain) brain homogenate in vitro for 2 h at 37^c. The samples were assayed for infectivity by analysis of scrapie incubation periods and their values compared to those obtained after extended (1–28 days) in vitro incubation. The scrapie incubation periods for scrapie agent-macrophage mixtures which had undergone extended in vitro incubation were longer than for mixtures assayed after a 2-hour exposure to the scrapie agent. The difference in scrapie incubation periods was more drΕmatic when residual scrapie was eliminated by washing the cells after the 2-hour exposure to brain homogenate. The scrapie incubation period also increased following in vitro incubation of ME7 in the absence of cells; however, the changes were less than those observed for scrapie agent-macrophage mixtures. In a culture in which cells had been destroyed by UV irradiation after exposure to the scrapie agent, there was more scrapie infectivity than in a comparable culture of untreated, scrapie-Εxposed macrophages. These results show that scrapie infectivity decreases with extended incubation of scrapie-exposed macrophages, and the data suggest that macrophages can inactivate the scrapie agent in vitro.

Scrapie brain homogenate was mixed with mouse peritoneal macrophages in vitro. After 2 h of incubation at 37°, a portion of the scrapie infectivity was associated with macrophages. In contrast, very little infectivity was associated with kidney cells that had been exposed to scrapie brain homogenate. After incubation of the scrapie brain homogenate-macrophage mixture at 4° rather than 37°, a reduced quantity of infectivity was associated with the macrophages. These results show that after in vitro incubation, the scrapie agent was associated with macrophages, and the data suggest that phagocytic activity was involved.