Emergence of classical BSE strain properties during serial passages of H-BSE in wild-type mice

Abstract

Background: Two distinct forms of atypical spongiform encephalopathies (H-BSE and L-BSE) have recently been identified in cattle. Transmission studies in several wild-type or transgenic mouse models showed that these forms were associated with two distinct major strains of infectious agents, which also differed from the unique strain that had been isolated from cases of classical BSE during the food-borne epizootic disease.

Methodology/principal findings: H-BSE was monitored during three serial passages in C57BL/6 mice. On second passage, most of the inoculated mice showed molecular features of the abnormal prion protein (PrP(d)) and brain lesions similar to those observed at first passage, but clearly distinct from those of classical BSE in this mouse model. These features were similarly maintained during a third passage. However, on second passage, some of the mice exhibited distinctly different molecular and lesion characteristics, reminiscent of classical BSE in C57Bl/6 mice. These similarities were confirmed on third passage from such mice, for which the same survival time was also observed as with classical BSE adapted to C57Bl/6 mice. Lymphotropism was rarely detected in mice with H-BSE features. In contrast, PrP(d) was detectable, on third passage, in the spleens of most mice exhibiting classical BSE features, the pattern being indistinguishable from that found in C57Bl/6 mice infected with classical BSE.

Conclusion/significance: Our data demonstrate the emergence of a prion strain with features similar to classical BSE during serial passages of H-BSE in wild-type mice. Such findings might help to explain the origin of the classical BSE epizootic disease, which could have originated from a putatively sporadic form of BSE.

Figure 1. Western blot analyses of brain PrP^res from mice infected with H-BSE.

Mice inoculated with with H-BSE (lanes 1–3 and 5–7 from isolates 01-2604 and 03-2095 respectively), at first (lanes 1, 5) or second passage (lanes 2–3 and 6–7), were compared to mice infected with classical BSE (lanes 4, 8) in panels A–C. At second passage, PrP^res exhibited either H-type features, from mice that died later (491 and 504 d.p.i. in lanes 2 and 6 respectively), or “C-BSE like” features (464 and 322 d.p.i. in lanes 3 and 7 respectively). The brain tissue equivalents loaded per lane are indicated (in tenth of mg). D - Glycoform proportions (means +/− standard deviations) of PrP^res from H-BSE (isolate 03-2095)-infected mice detected using Sha31 antibody. Mice exhibited PrP^res with a molecular mass either similar to that of H-BSE (full squares) or to classical BSE (full diamonds). E - PrP^d extracted in the absence of protease digestion and deglycosylated by PNGase treatment from H-BSE infected mice at first (lane 1) or second passage (lanes 2-3 from mice that died at 497 d.p.i. and 464 d.p.i. respectively), compared to a classical BSE control in lane 4 (0.3 mg brain tissue equivalent per lane). Full-length PrP^d (FL) and C1 and C2 fragments are indicated. Panels A and B were revealed by monoclonal antibodies Sha31 and 12B2 respectively and panels C and E by HRP-labelled SAF84 antibody. Bars to the left indicate the 29.0, 20.1 and 14.3 kDa marker positions.

Figure 2. Western blot analyses of PrP^res in the spleens of mice infected with H-BSE.

A - Detection of PrP^res from spleens of mice inoculated with H-BSE isolates 01-2604 (lanes 1–3) or 03-2095 (lanes 4–9) at first passage. B - Detection of PrP^res from spleens of mice inoculated with H-BSE at third passage in mice inoculated with a brain homogenate with “C-BSE like” PrP^res (lanes 4–8). PrP^res from the brain of one of these mice (lanes 3, 9) and from brains of C506M3 scrapie (lanes 1, 11) or classical BSE (lanes 2, 10) controls are shown for comparison. C - PrP^res from spleens of mice infected with H-BSE at first passage (lanes 2, 4)(isolates 03-1928 and 03-2095), second passage in a mouse with “C-BSE like” PrP^res (lane 6) and third passage from mice inoculated with a brain homogenate with “C-BSE like” PrP^res (lanes 8, 10). PrP^res from spleens of classical BSE (lanes 3, 5, 7, 9) and C506M3 (lanes 1, 11) controls are shown. Panels A and B were revealed by monoclonal antibody Sha31 and panel C by HRP-labelled SAF84 antibody Bars to the left indicate the 29.0 and 20.1 kDa marker positions in panels A and B or the 20.1 and 14.3 kDa marker positions in panels C and D.

Figure 3. Histopathological analyses of mice infected with H-BSE at second passage.

Brain distribution of the disease-associated prion protein (PrP^d) observed in the brain of C57Bl/6 mice infected with 3 isolates of H-BSE, at second passage. The green color gives the schematic representation of PrP^d, the dots symbolize the plaque type deposits. Plaques of amyloid nature as revealed by a Red Congo staining observed under polarized light (A2 & D3) and made of PrPd deposits (A1, D2, E1, E2) (black staining after IHC) were similarly detected in each H- BSE transmission studies and remarkably this amyloid type of PrP^d deposition was the predominant histopathological features typical of H-BSE. In two out of three second passage experiments, another sub-group of mice was clearly identified as showing a different PrP^d brain mapping. This sub-group showed much more brain areas accumulating PrP^d with a granular type of staining (B, D1), reminding most of the features seen in the case of the classical BSE features (F1, F3) of which also typical spongiform changes in the cochlear nucleus (F2).

Figure 4. Histopathological analyses of mice infected with H-BSE at third passage.

Brain distribution of the disease-associated prion protein (PrP^d) observed in the brain of C57Bl/6 mice infected with H-BSE, at third passage from a mouse with H-type features (A) or with “C-BSE like” features (B). The green color gives the schematic representation of PrP^d, the dots symbolize the plaque type deposits. In right panels, pictures of some characteristics PrP^d deposits for 3^rd passage of H-BSE from mouse with H-type features in thalamus as plaques solely (A1) or from mouse with C-type features in the cortex (B1). The cochlear nucleus showed spongiform lesions (B2 compared to A2) and granular PrP^d brain deposits (dark deposits of DAB intensified using NiCl2) (B3 compared to A3) in the C-type solely. Scale bars: 50 µm for all panels and 10 µm for the plaque focus in panel A1.