In the current study, transmission of disease by inoculation of G131V human brain was also suspected as 11 of 11 tg66 recipient mice showed suggestive clinical signs and convincing PrPSc deposits in brain tissue between 531 and 784 dpi. However, these brains were not positive for PrPSc by immunoblot and did not show PrP amyloid seeding activity by RT-QuIC assay. One additional mutation Q227X, was also tested in the present study and was negative by all parameters measured so far in first passage tg66 mice. Second passages of tg66 mice inoculated with both the G131V and Q227X brain tissue are in early stages, and no positive conclusions can be drawn at this time.