Isolation from Cattle of a Prion Strain Distinct from That Causing Bovine Spongiform Encephalopathy

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrP(res) of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission

Lesion Profiles in tgBov Mice Infected with H-Type or BSE Agents

<p>Mean scores (± SEM) reflecting the intensity of vacuolation are shown for H-type (no. 1 and no. 3, triangles) and BSE (no. 1 and no. 3, squares). The nine grey-matter areas used to construct the profile are as follows: dorsal medulla (1); cerebellar cortex (2); superior colliculus (3); hypothalamus (4); medial thalamus (5); hippocampus (6); septum (7); medial cerebral cortex at the level of the thalamus (8); and medial cerebral cortex at the level of the septum (9).</p

Survival Time in Transgenic Mice Infected with H-Type and BSE-Type Agents

<p>Mean survival times (days ± SEM) upon primary transmission are shown for tgBov (grey circles) and tgOv (black diamonds) mice inoculated with H-type cases, BSE, and related isolates (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020112#ppat-0020112-t001" target="_blank">Table 1</a>). The intervals between the incubation times on each line are significantly different for H-type and BSE agents (<i>p</i> < 0.0002, Fisher test).</p

Comparison of H-Type and SSit Isolate Features upon Transmission to tgOv Mice

<div><p>(A and B) Western blot analysis of PrP^res in the brains and spleens of tgOv mice infected with H-type or SSit isolates at first (lanes 2–4) and second passage (lanes 5–11). H-type PrP^res shows a distinct pattern in the brain (Br) compared to SSit. The apparent molecular mass of SSit PrP^res is higher than that of H-type or sheep BSE (shBSE), as shown after PNGase treatment (B). Note also that PrP^res is detected in the spleen (Sp) of SSit- but not of H-type–infected mice. Tissue equivalent loaded: 1.5 mg in lanes 2–4; 0.04 mg in lane 5; 0.5 mg in lanes 6–7; 2 mg in lane 8; 0.01 mg in lane 9; 0.1 mg in lanes 10–11. MM, molecular markers.</p><p>(C) Ratio of high- and low-molecular-mass PrP^res glycoforms in the brain of tgOv mice infected with H-type or SSit isolates (data plotted as mean ± SEM). One H-type isolate (no. 2) is represented as orange triangle. SSit isolates are represented as circles (SSit no. 5, red; no. 7, brown; no. 8, yellow). Secondary transmissions are represented by unfilled symbols of the same colour. Note the stably distinct glycoform ratios between H-type and SSit agents upon serial passage.</p><p>(D) Regional distribution of PrP^res in the brain of tgOv mice infected with H-type or SSit isolates. Histoblots of representative coronal sections of tgOv mouse brains at the levels of the hippocampus are shown. The distribution of H-type–associated PrP^res deposits was different from that of SSit in regions such as the alveus of the hippocampus, the corpus callosum, the pretectal nuclei, the cortex, and the ventromedial thalamus. Note that the size of PrP^res deposits markedly differed between the two types of isolates.</p></div

Western Blot Analysis of PrP^res in the Brains of Transgenic Mice Infected with H-Type or BSE-Type Agents

<div><p>(A) Primary or secondary (lane 6) transmission to tgBov mice. BSE-type inocula include cattle BSE no. 3 (lanes 3 and 7), sheep BSE ARQ no. 1 and ARR no. 1 (lanes 8 and 9), goat BSE (lane 10), and vCJD (lane 11). The PrP^res profiles of both H-type and BSE agents in cattle (lanes 2 and 3) are essentially similar to those in tgBov mice (lanes 4–11). Brain tissue equivalent loaded: 2.5 mg in lane 2; 0.15 mg in lane 3; 0.5 mg in lanes 4–12. MI, mock-infected brain; MM, molecular markers.</p><p>(B) Primary transmission to tgOv mice. H-type agent shows a distinct PrP^res pattern in the brain (Br) compared to BSE agents (lane 9, BSE no. 3; lane 10, goat BSE; lane 11, vCJD). Note the lack of PrP^res signal in the spleen (Sp) of H-type–infected mice (lane 7), unlike that in BSE-infected mice (lane 8). Brain or spleen tissue equivalent loaded: 3 mg in lane 2; 0.15 mg in lane 3; 0.5 mg in lanes 4–6; 2 mg in lanes 7–12.</p><p>(C and D) Ratio of high- and low-molecular-mass PrP^res glycoforms in the brains of tgBov (C) and tgOv (D) mice following challenge with H-type or BSE agents (data plotted as means ± SEM). H-type isolates are represented as triangles (no. 1, blue; no. 2, orange; no. 3, pink; and no. 5, black) and BSE agents as squares (BSE no. 3, red; sheep BSE ARQ no. 1, grey; sheep BSE ARR no. 1, yellow; goat BSE, brown; and vCJD, light blue). Secondary transmissions are represented by unfilled symbols of the same colour. Note the strikingly distinct glycoform ratio between H-type and BSE groups in both mouse lines, as reported in cattle [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020112#ppat-0020112-b009" target="_blank">9</a>].</p></div