Transmission studies of chronic wasting disease to transgenic mice overexpressing human prion protein using the RT-QuIC assay

Abstract

Chronic wasting disease (CWD) is a fatal prion disease which infects deer, elk and moose. CWD was first described as a wasting syndrome in captive deer in Colorado and Wyoming wildlife facilities from 1967 to 1979. Currently, CWD has been reported in 26 states of the USA, three Canadian provinces, South Korea, Norway and Finland. Since human consumption of cervids is common, it is critical to determine if CWD can infect humans. Published research, including epidemiologic studies and transmission studies using animal models, including transgenic mice that express human prion protein, have suggested existence of a strong species barrier between cervid CWD and humans. In the current study, we tested CWD transmission into two additional strains of transgenic mice (tg66 and tgRM). These mice over-express human prion protein at high levels and are highly sensitive to infection by human-tropic prions. One hundred and eight mice were inoculated intracerebrally with three different sources of CWD. After long periods of observation, brain tissues from CWD-inoculated mice were screened for evidence of prion infection by RT-QuIC, immunohistochemistry (IHC) and immunoblot. No IHC or immunoblot evidence was found to suggest transmission had occurred, and most mice were negative by RT-QuIC assay. However, four mice with inconsistent positive RT-QuIC reactions were detected. The seeding activity detected in these mice may represent a low level of CWD agent, suggesting a possible transfer of CWD infection. Alternatively, these results might be due to false positive reactions or residual CWD inoculum.

Figure 1

RT-QuIC analysis of uninoculated and sCJD inoculated transgenic mice. All brain samples were tested at a 10⁻³ dilution. In A–F, four independent wells from each mouse are represented as individual curves with unique symbols. A–E Data from five different uninoculated tg66 and tgRM mice tested with Ha rPrP substrate. F An example of an uninoculated tg66 mouse tested with BV rPrP substrate. G Ha rPrP RT-QuIC data from several replicate trays of uninoculated transgenic mice (left side) and sCJD-positive control (right side) transgenic mice at the 25-h timepoint. Each point represents measured fluorescence in a single test well. Different tray numbers and different mice are indicated below the x-axis. A horizontal dashed line is shown at 10% florescence (27 standard deviations above baseline florescence) that clearly separated negative and positive reactions.

Figure 2

RT-QuIC analysis for PrP amyloid seeding activity in brains from sCJD-infected tg66 mice or CWD-positive elk brain. A–D The sCJD-infected tg66 brain (B946-1) and panels E-H show the CWD-positive elk brain pool (Elk-2). In A and E, a ten-fold dilution series is shown. In these panels, each curve represents and average fluorescence of four replicate wells per dilution. Dilutions are indicated to the right of each panel. B–D and F–H Detailed data from individual dilutions (indicated in the panel titles). In these panels, four wells were tested at each dilution and each curve represents data from an individual well. Note how the ratio of wells with amyloid seeding activity decreases at the sample becomes more dilute (for example, compare F–H). For all the data shown, RT-QuIC was performed using Ha rPrP 90–231 substrate.

Figure 3

RT-QuIC data from sCJD-infected, uninfected and indeterminate CWD-inoculated tg66 mice. Additional RT-QuIC analysis was performed on four CWD-inoculated mice that gave positive amyloid seeding activity results on our initial screening runs to assess reproducibility of the data. Each point represents the normalized fluorescence value for an individual well measured at 25 h of RT-QuIC reaction time. Results from 3 to 4 independent assays testing 4–16 wells per mouse per assay have been pooled. The total number of wells tested for each mouse and the number of positive and negative control wells present on the same trays is provided below the x-axis. Statistical analysis was performed comparing data from individual mice to the uninfected control mice using Fisher’s exact test, *** indicates p < 0.0001, ** indicates p = 0.0013.

Figure 4

Immunohistochemical staining and neuropathology in sCJD and CWD-inoculated tg66 mice. Whole brain sections stained with anti-prion protein antibody b3F4 are shown in A, E and I. The small rectangle shown in the whole brain sections depicts the region of thalamus shown at higher magnification on the right. Thalamus panels were stained with b3F4, H&E or GFAP (shown above each column). Tissues were stained with the anti-PrP antibody to detect PrP deposition, by H&E to look for spongiosis/vacuolation and general neuropathology, and anti-GFAP was used to detect activated astrocytes. A–D Are from a tg66 mouse infected with sCJD, E–H are from a normal, aged tg66 mouse and I–L are from an aged CWD-inoculated tg66 mouse. No pathology, spongiform lesions, or excessive astroglial activation was observed in any of the uninoculated or CWD-inoculated tg66 brains. PrPsen could be seen in all brains as a smooth brown blush using anti-PrP antibody b3F4. The scale bar in A is 1 mm and applies to A, E and I. The scale bar in L is 50 µm and applies to B–D, F–H, and J–L.

Figure 5

Immunohistochemical staining and neuropathology in sCJD and CWD-inoculated tgRM mice. Whole brain sections stained with anti-prion protein antibody b3F4 are shown in A, E and I. The small rectangle shown in the whole brain sections depicts the region of thalamus shown at higher magnification on the right. Thalamus panels were stained with b3F4, H&E or GFAP (shown above each column). Tissues were stained with the anti-PrP antibody to detect PrP deposition, by H&E to look for spongiosis/vacuolation and general neuropathology, and anti-GFAP was used to detect activated astrocytes. A–D Are from a tgRM mouse infected with sCJD, E–H are from a normal, aged tgRM mouse and I–L are from an aged CWD-inoculated tgRM mouse. No pathology, spongiform lesions, or excessive astroglial activation was observed in any of the uninoculated or CWD-inoculated tgRM brains. The PrPsen background staining seen in tg66 mice is not present in tgRM mice using the same staining methods. This is likely due to the lower PrPsen expression in tgRM mice. The scale bar in A is 1 mm and applies to A, E and I. The scale bar in L is 50 µm and applies to B–D, F–H and J–L.

Figure 6

Immunoblot screening for PrPres in CWD-inoculated tg66 and tgRM mice. Brains from CWD-inoculated transgenic mice were treated with proteinase K (PK) or untreated and processed for immunoblot as described in the methods. A, B Results without PTA precipitation, C, D used PTA precipitation to concentrate potential low levels of PrPres. CWD inoculum and mouse numbers are shown across the top of each immunoblot. PK and PTA treatment status are shown across the bottom. A (tg66 mice): lane 1, tg66 mouse infected with vCJD; lanes 2, 3 uninoculated tg66 mouse; lanes 4–11 show several tg66 CWD-inoculated mice, including two RT-QuIC suspect mice (bolded). B (tgRM mice): lane 1, tg66 mouse infected with vCJD; lanes 2, 3 uninoculated tgRM mouse; lanes 4–11 show several tgRM CWD-inoculated mice. For both A and B, lane 1 was loaded with 0.36 mg tissue equivalents (te), lanes 2 and 4–11 were loaded with 0.72 mg te each and lane 3 was loaded with 0.04 mg te. C, D (PTA precipitation of tg66 mice): lane 1, tg66 mouse infected with vCJD; lanes 3, 4 uninoculated, age-matched tg66 mice; lanes 5–10 in C and 5–9 in D show several tg66 CWD-inoculated mice, including two RT-QuIC suspect mice (bolded). Lane 10 in D shows PTA precipitated PrPsen from an uninfected tg66 mouse. For C and D, lane 1 was loaded with 0.24 mg te, lanes 2–10 in C and lanes 2–9 in D were loaded with 22.7 mg te and lane 10 in D was loaded with 1.35 mg te. The immunoblots were probed with anti-PrP antibody 3F4 and developed using a femto detection system.