In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv^109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv^109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv^109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrP^Sc (PrP^res) in the same brain regions. Despite the low PrP^res levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 10^8,4 i.c. ID₅₀ infectious units per gram. Bv^109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv^109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv^109ICWD showed unique characteristics of “virulence”, low PrP^res accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrP^Sc, neurodegeneration and infectivity. Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids and is expanding increasingly in the USA and Canada. Animal models are of key importance in the study of prion diseases but their development for CWD has long been hampered by its very inefficient transmission to wild-type mice. Significant progress was made following the generation of transgenic mice over-expressing cervid PrP. Here we show that the bank vole (Myodes glareolus), a wild rodent species that we demonstrated to be susceptible to many animal and human prion diseases, is also very susceptible to CWD from elk, mule deer and white-tailed deer. Adaptation of CWD to bank vole led to the isolation of a prion strain with peculiar characteristics: unprecedented short incubation and survival times, respectively of 25–28 and ∼35 days, low PrP^Sc levels compared with other vole-adapted prion strains and high infectious titre. These features were all faithfully maintained upon the generation of this strain in vitro by protein misfolding cyclic amplification. The development of a model for prion diseases that led to disease in less than one month accumulating high infectious titres but low PrP^Sc levels, represents a significant tool for investigating the still unclear relationship between PrP^Sc, neurodegeneration and infectivity in prion diseases.

The bank vole is a rodent susceptible to different prion strains from humans and various animal species. We analyzed the transmission features of different prions in a panel of seven rodent species which showed various degrees of phylogenetic affinity and specific prion protein (PrP) sequence divergences in order to investigate the basis of vole susceptibility in comparison to other rodent models. At first, we found a differential susceptibility of bank and field voles compared to C57Bl/6 and wood mice. Voles showed high susceptibility to sheep scrapie but were resistant to bovine spongiform encephalopathy, whereas C57Bl/6 and wood mice displayed opposite features. Infection with mouse-adapted scrapie 139A was faster in voles than in C57Bl/6 and wood mice. Moreover, a glycoprofile change was observed in voles, which was reverted upon back passage to mice. All strains replicated much faster in voles than in mice after adapting to the new species. PrP sequence comparison indicated a correlation between the transmission patterns and amino acids at positions 154 and 169 (Y and S in mice, N and N in voles). This correlation was confirmed when inoculating three additional rodent species: gerbils, spiny mice and oldfield mice with sheep scrapie and 139A. These rodents were chosen because oldfield mice do have the 154N and 169N substitutions, whereas gerbil and spiny mice do not have them. Our results suggest that PrP residues 154 and 169 drive the susceptibility, molecular phenotype and replication rate of prion strains in rodents. This might have implications for the assessment of host range and molecular traceability of prion strains, as well as for the development of improved animal models for prion diseases. Prions are unconventional infectious agents that cause fatal neurodegenerative diseases in animals and humans. A pathological form of the cellular prion protein (PrP^C), named PrP^Sc, appears to be the major or the sole component of prions. These agents are transmitted by inducing the conversion of host PrP^C into PrP^Sc that accumulates in the brain of affected individuals. Different factors are believed to modulate such events, which explains the variable transmission efficiency observed under inter-species experimental inoculation. These factors are still fairly unknown, although evidence exists that some kind of structural compatibility between PrP^Sc of the infectious inoculum and PrP^C of the host has a role in making transmission more or less efficient. We investigated the transmission of prions to different rodents and showed that specific amino acid substitutions (Y154N and S169N) in the prion protein are major determinants of susceptibility to prions. In particular, we showed that these specific variations i) direct the transmission rate of prions between different species in a way that is dependent on the prion strain, ii) affect the molecular characteristics of prions, and iii) influence their replication efficiency.

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.

Variable Protease-Sensitive Prionopathy

Bovine Spongiform Encephalopathy (BSE) is the only animal prion which has been recognized as a zoonotic agent so far. The identification of BSE in two goats raised the need to reliably identify BSE in small ruminants. However, our understanding of scrapie strain diversity in small ruminants remains ill-defined, thus limiting the accuracy of BSE surveillance and spreading fear that BSE might lurk unrecognized in goats. We investigated prion strain diversity in a large panel of European goats by a novel experimental approach that, instead of assessing the neuropathological profile after serial transmissions in a single animal model, was based on the direct interaction of prion isolates with several recipient rodent models expressing small ruminants or heterologous prion proteins. The findings show that the biological properties of scrapie isolates display different patterns of geographical distribution in Europe and suggest that goat BSE could be reliably discriminated from a wide range of biologically and geographically diverse goat prion isolates. Finally, most field prion isolates showed composite strain features, with discrete strain components or sub-strains being present in different proportions in individual goats or tissues. This has important implications for understanding the nature and evolution of scrapie strains and their transmissibility to other species, including humans.

Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrP^Sc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrP^Sc biochemical characteristics and if it was possible to univocally identify strains through PrP^Sc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived) and analyzed PrP^Sc from individual voles by epitope mapping of protease resistant core of PrP^Sc (PrP^res) and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrP^Sc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed.