Conceived and designed the experiments: UA RN GD HPL. Performed the experiments: UA RN GD MADB MC BC EE OW GV. Analyzed the data: UA RN GD HPL. Contributed reagents/materials/analysis tools: GDG. Wrote the paper: UA RN HPL.
The authors have declared that no competing interests exist.
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 (PrPC), named PrPSc, appears to be the major or the sole component of prions. These agents are transmitted by inducing the conversion of host PrPC into PrPSc 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 PrPSc of the infectious inoculum and PrPC 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.
The conversion of the cellular prion protein (PrPC) into an abnormally-folded isoform (PrPSc) that accumulates in the brain of affected individuals represents the key feature of transmissible spongiform encephalopathies (TSEs), or prion diseases
Experimental animals are of paramount importance for the study of TSEs. However, very long incubation periods or even unsuccessful transmissions are observed when a given model is challenged with prions from a different species. Prion transmission to a new species is in fact limited by a phenomenon known as “species barrier”
Early studies argued that the main factor influencing interspecies transmission resides in the homology degree of the amino acid sequence of PrP between the donor and recipient species
Recently, we reported that the interspecies transmission of prions from humans to bank voles (
Studies of transmission barrier are important for elucidating the basis of prions replication and acquiring knowledge to decipher the risk of interspecies transmission. The availability of animal models susceptible to different prion strains is of crucial relevance for such kind of studies.
We recently showed that the bank vole is very susceptible to TSEs
Transmission studies were first set up in bank and field voles in comparison to C57Bl/6 and wood mice. Concerning natural scrapie (SS3), the results of primary transmission to bank voles and C57Bl/6 mice were previously reported
One hundred per cent of bank and field voles developed obvious clinical signs and were sacrificed after short survival times following inoculation of natural scrapie (
Recipient species | Inoculum | Primary transmission | Second passage | |||||
Clin. signs (+) | Pathol. (+) | PrPSc (+) | Survival time (days±SD) | Transm. rate (%) | PrPSc (+)/inoculated | Survival time (days±SD) | ||
Bank voles | SS3 |
9/9 | 9/9 | 9/9 | 199±28 | 100 | 7/7 | 92±14 |
BSE | 0/6 | 0/4 | 0/6 | >1044 |
0 | 6/6 | 483±85 | |
Field voles | SS3 | 8/8 | 8/8 | 8/8 | 259±53 | 100 | n.d. | n.d. |
BSE | 0/10 | 0/8 | 0/5 | >707 |
0 | n.d. | n.d. | |
Wood mice | SS3 | 0/7 | 0/7 | 0/7 | >1341 |
0 | n.d | n.d |
BSE | 9/9 | 8/8 | 9/9 | 720±38 | 100 | n.d. | n.d. | |
C57Bl/6 mice | SS3 |
0/9 | 3/9 | 3/9 | 567±149° | 33° | 16/16 | 233±9 |
BSE | 4/7 | 6/7 | 6/7 | 631±25° | 86° | 10/10 | 194±2 |
Results of the transmission of SS3 to bank voles and C57Bl/6 mice were previously reported
When no PrPSc-positive animal was found in the group, survival time is shown as longer (>) than the survival time of the last sacrificed/dead animal.
Survival times and transmission rates were calculated only on animals showing PrPSc accumulation.
In contrast, the inoculation of natural scrapie in C57Bl/6 and wood mice produced very long survival times without overtly suggestive signs of prion disease. C57Bl/6 mice rarely showed subtle and equivocal signs such as nervousness and hyper- reactivity, followed by apathy.
PrPSc brain accumulation was detected by Western blot in 100% of bank and field voles whereas it was not detected in wood mice and in only three out of nine C57Bl/6 mice (
The reverse situation was apparent following the inoculation of BSE. Neither of the two vole species showed clinical signs or PrPSc accumulation during their lifetime. This is in contrast to C57Bl/6 and wood mice which showed overt neurological signs characterized by hyper-activity/reactivity and followed by hind limb incoordination, hunched posture and apathy. Survival times were rather long in both mice species, but the transmission rate was high (
Western blot showed that the apparent molecular weight (MW) of proteinase K-treated PrPSc from BSE and natural scrapie was maintained after transmission to rodents. Indeed, the unglycosylated isoform of PrPSc from BSE was ∼1 kDa lower than scrapie (
(A) bank voles, (B) field voles, (C) wood mice and (D) C57Bl/6 mice.
Second passages were performed on C57Bl/6 mice and bank voles, which were considered representative of the two rodent groups. They were successful in all inoculated animals (
Immunoblot of PrPSc from the second passage of BSE to bank voles (lane 1), the primary transmission of 139A to bank voles, field voles, oldfield mice and gerbils (lanes 2, 3, 4, 6), the transmission of 139A to C57Bl/6 mice (lanes 7, 8) and the back passage of vole-adapted 139A to C57Bl/6 mice (lane 5).
The third passages of SS3 and BSE were carried out in order to investigate their adaptation to bank voles and C57Bl/6 mice (
Mean survival times±SD are reported at the top of each bar.
Both SS3 and BSE adapted to voles as very rapid strains, while longer survival times were observed after their adaptation to C57Bl/6 mice (
The primary transmission results of natural scrapie and BSE suggested a differential susceptibility of the two vole species on one side, and of C57Bl/6 and wood mice on the other. Bank voles, field voles and wood mice were inoculated with the 139A strain and the transmission characteristics compared to those observed in C57Bl/6 mice in order to investigate if such a pattern of susceptibility was maintained even after the inoculation of a well characterized mouse-adapted scrapie strain.
139A was transmitted very efficiently (100% transmission rate) to all species. All inoculated animals showed clinical signs and revealed spongiform degeneration and PrPSc accumulation in their brain (
Recipient species | Primary transmission | Second passage | |||||
Clin. signs (+) | Pathol. (+) | PrPSc (+) | Survival time (days±SD) | Transm. rate | PrPSc (+)/inoculated | Survival time (days±SD) | |
Bank voles | 19/19 | 19/19 | 19/19 | 134±14 | 100 | 7/7 | 75±11 |
Field voles | 8/8 | 8/8 | 8/8 | 126±10 | 100 | 7/7 | 87±7 |
Wood mice | 11/11 | 11/11 | 11/11 | 191±25 | 100 | 9/9 | 149±23 |
C57Bl/6 mice | 9/9 | 159±3 |
The transmission of 139A to C57Bl/6 mice is shown in comparison with the second passage to the other species.
Strikingly, both vole species showed shorter survival times than C57Bl/6 mice which is the species to which that strain is adapted. Wood mice showed the longest survival times among the four species (
Spongiosis was widespread in the brain of all species with the exception of the cerebellar cortex. Both granular and molecular layers of cerebellar cortex were targeted by moderate/high vacuolar degeneration in C57Bl/6 and wood mice, while spongiosis was only occasional and confined to the granular layer in field and bank voles (
(A) bank voles, (B) field voles, (C) oldfield mice, (D) wood mice, (E) gerbils and (F) C57Bl/6 mice. Spongiosis is widespread in both molecular and granular layers of wood mice, gerbils and C57Bl/6 mice. In contrast, the cerebellar cortex is quite completely spared in the other three rodent species in which occasional vacuoles were observed only in the granular layer. Bar = 50 µm.
Interestingly, the molecular analysis of PrPSc provided further evidence of the differences in the transmission features of prions between voles and mice. As a matter of fact, the typical 139A glycoprofile in mice, monoglycosylated>diglycosylated>unglycosylated, was faithfully maintained in wood mice, while it clearly changed to a diglycosylated>monoglycosylated>unglycosylated pattern in voles (
Second passage of 139A was carried out in the three rodent species under investigation. Survival times were very short in both vole species, while in wood mice they were rather long and similar to those observed in C57Bl/6 mice (
The adaptation of 139A confirmed the very short survival times of vole-adapted strains, which were already observed with SS3 and BSE. The hypothesis of a high expression level of PrPC which accounted for these findings, was ruled out by Western-blot and Histo-blot analyses, because they did not show any significant differences either in the distribution or in the level of PrPC expression in the brain of bank voles, field voles, wood mice and C57Bl/6 mice (data not shown).
139A was fully adapted and stabilized in bank voles with the third passage and subsequently inoculated back into C56Bl/6 mice in order to investigate if the novel PrPSc glycoprofile observed in voles inoculated with 139A could have been considered as the emergence of a different strain with a new stable molecular signature. The third passage of 139A to bank voles produced the same survival time (76±8 d.p.i.) and PrPSc characteristics (data not shown) as the second passage. This suggested that the strain had already been adapted to the new host at the second passage.
After inoculation, all C57Bl/6 mice (n = 20) developed the disease showing spongiform change and PrPSc accumulation in their brain. Survival times were long (463±62 d.p.i.), suggestive of the existence of a transmission barrier also during the transmission from voles to mice, the species to which 139A was originally adapted. Worth mentioning is the fact that the molecular characteristics of PrPSc reverted to that of the original mouse inoculum (
The comparison of PrP sequences of the bank vole, field vole, wood mouse and laboratory mouse displayed a high homology degree, although a number of substitutions were found in the N-terminal cleaved signal peptide and in the C-terminal signal sequence that is also cleaved when the GPI-anchor is added. Sequence comparison showed relevant amino acid substitutions at only five positions (
The sequence numbers of C57Bl/6 mouse (
The first substitution, G89S, was at the N-terminus non-structured tail of PrP and was observed only in the field vole. The second replacement, L108M, was in the N-terminal disordered tail and is known to influence the susceptibility of both voles
Two substitutions were found in the structured C-terminal domain. A replacement Y154N was found in the loop region between the first α-helix and the second β-strand, while a substitution S to S169N was in the loop between the second β-strand and the second α–elix (
Different secondary structure elements are drawn in different colours (purple: α-helix, yellow: β-sheet, green: loop).
We especially focused our attention on the two variations observed in the structured C-terminal domain of PrP, which are located into regions that supposedly contribute to the species barrier because they apparently function as selective protein-protein interaction sites or are involved in the specificity of intermolecular interactions
In order to test the hypothesis of the role of Y154N and S169N substitutions in influencing the transmission and phenotype characteristics of prions to rodents, we analyzed the PrP sequence of other rodents frequently bred under laboratory conditions and hence selected for transmission studies three additional species: the oldfield mouse, the Mongolian gerbil and the spiny mouse. They were chosen because oldfield mice showed Y154N and S169N substitutions, whereas gerbil and spiny mice did not show them (
All species with Y154–S169 residues (gerbil, C57Bl/6, wood and spiny mouse) fall in the family of Muridae, while species with 154N–169N (oldfiled mouse, bank and field vole) fall in the family of Cricetidae.
Groups of oldfield mice, spiny mice and gerbils were challenged with the same inocula of 139A and natural scrapie used in previous transmissions. Following inoculation of both scrapie sources, oldfield mice developed the disease with short survival times, comparable to those of voles, while gerbils showed a very inefficient transmission of natural scrapie and long survival times after inoculation of 139A (
Recipient species | Inoculum | Clin. signs (+) | Pathol. (+) | PrPSc (+) | Surv. time (days±SD) | Transm. rate |
Oldfield mice | 139A | 10/10 | 8/8 | 9/9 | 151±8 | 100 |
SS3 | 12/12 | 12/12 | 11/11 | 206±23 | 100 | |
Spiny mice | 139A |
0/10 | 0/1° | 0/1° | >1800 | 0 |
SS3 |
0/7 | 0/7 | 0/7 | >1300 | 0 | |
Mongolian gerbils | 139A | 5/5 | 5/5 | 5/5 | 238±12 | 100 |
SS3 |
1/5 | 1/1 | 1/1 | 1032; >1300 | 20 |
Experiment in progress. In these cases, the time elapsed from inoculation to writing is reported as survival time.
No pathological changes or PrPSc accumulation were detected in the brain of one spiny mouse which was sacrificed 1021 days after inoculation for intercurrent disease (tumour).
In accordance with the overall data, phenotypic analysis of this second set of transmissions by brain histopathology and molecular analysis of PrPSc revealed characteristics which paralleled those observed in voles, wood mice and laboratory mice. A severe vacuolar degeneration of molecular and granular layers of the cerebellum was evident in gerbils, while the molecular layer was completely spared in oldfield mice (
Finally, since we observed that scrapie and BSE adapted to bank voles as much faster strains than in mice, we checked if this also applied to oldfield mice by setting up the second passage of SS3 in that species. The survival time of SS3 in oldfield mice was indeed short (103±11 d.p.i.) and comparable to that observed in bank voles.
We showed that the rodents under investigation can be subdivided into three groups. The first included voles and the oldfield mouse and was characterized by: i) high susceptibility to scrapie, ii) low susceptibility to BSE, iii) extremely short incubation times with adapted strains and iv) change in the 139A glycoprofile. The second group comprised C57Bl/6 mice, wood mice and gerbils and displayed: i) low susceptibility to scrapie, ii) relatively high susceptibility to BSE, iii) longer incubation times with adapted strains and iv) no change in the 139A glycoprofile. The third group included only spiny mice which showed a distinctive resistance to prions.
These findings were consistent with the inefficient transmission of natural scrapie to wild type mice reported by several authors
PrP sequence comparison indicated that Y154N and S169N correlated with the different transmission patterns observed. Overall, species with Y154–S169 were resistant to scrapie, permissive to BSE and reproduced a mouse-like phenotype when infected with 139A, while species with 154N–169N displayed rather opposite features.
The inverted susceptibility of rodents to scrapie and BSE underlined the role of strains in the transmission barrier: amino acid exchanges could either enhance or reduce the efficiency of transmission, depending on the prion strain. In particular, we showed that Y154N–S169N exchanges, which appeared to confer in vole-related species a high susceptibility to scrapie, had the opposite effect with BSE. This is concordant with
The change in the 139A glycoprofile further corroborated the distinction between vole- and mouse-related species. It is known that the PrPSc glycoform pattern is not necessarily preserved upon interspecies transmission
Piening et al
On this basis, it is tempting to speculate that voles allow a particularly efficient adaptation and/or rapid replication of prions, as suggested also by the unusually short incubation times of adapted strains. This latter was a striking feature of bank vole, given that almost all vole-adapted prions showed survival times ranging from ∼35 to 130 d.p.i., irrespective of whether they derived from humans, cattle, sheep, deer, mice, or hamsters (
The Syrian hamster model has represented a major advance in prion research owing to the extremely short incubation period of the hamster-adapted strain 263K
The molecular basis of interspecies transmission and adaptation of prions are unknown. Nevertheless, evidence suggests that the PrP sequence of the recipient species acts by dictating the range of possible PrP conformations and hence conditioning the susceptibility to different prion strains
Positions 154 and 169 are quite variable among mammalian PrPs. Human and bovine sequences are 154H–169S, sheep and goat 154Y–169S, elk and deer 154Y–169N. Considering the strain-related effect of variations at these positions, it could be speculated that such differences may account for the apparent limitation of prion interspecies transmission observed among humans, cervids and small ruminants. Actually, the only TSE proven to have crossed a species barrier naturally is BSE, which transmitted from cattle to humans; two species that share the same amino acids at positions 154 and 169.
In the three-dimensional representation of mouse PrPC, residues 154Y and 169S, corresponding to 154N and 169N of the vole prion protein, are exposed on the protein's surface (
The distinction between vole- and mouse-related species inferred by transmission studies is paralleled by the taxonomy, which classifies voles and oldfield mice in the family of Cricetidae, while the remaining species in that of Muridae
Finally, our study showed that the range of rodent models with improved susceptibility to TSEs is wider than it has appeared in studies up to date. Moreover, the high susceptibility of voles and oldfield mice to TSEs gave rise to questions about the possible role of wild rodents in the natural spread of animal TSEs suggesting an intriguing field for epidemiological investigations.
Bank voles (
Subjects were individually identified by passive integrated transponders, inoculated when weanlings (40–60 days) and kept in groups of two-four individuals per cage.
Scrapie-infected brain tissue (SS3) was obtained from the thalamus of a naturally-affected sheep of Sarda breed from Tuscany, which carried the AA136RR154QQ171 PrP genotype. The mouse-adapted scrapie strain 139A was kindly provided by Prof. M. Pocchiari (Istituto Superiore di Sanità). The BSE inoculum was prepared from the
Animals were anaesthetized with ketamine and inoculated intracerebrally (i.c.) into the left hemisphere with 20 µl brain homogenate. Beginning one month after inoculation, animals were examined twice per week until the appearance of clinical symptoms, and then examined daily. We measured the survival time instead of the incubation time because of the differences among species in the clinical phenotype of the disease. Diseased animals were sacrificed with carbon dioxide at the terminal stage of disease but before neurological impairment was such as to compromise welfare and, especially, adequate drinking and feeding. Survival time was calculated as the interval between inoculation and sacrifice or death.
After collection at sacrifice, each brain was cut parasagitally into two parts. The smaller one was stored at −80°C for biochemical studies. The other part was fixed in formalin for histology and immunohistochemistry analysis as described previously
Genomic DNA was extracted from frozen brain samples using standard procedures. The coding region of the PrP gene from each species was amplified from 100 ng of genomic DNA using the polymerase chain reaction (PCR). PCR reactions were performed with either MoPrP5 (
The spiny mouse, the oldfield mouse and the bank vole PrP coding regions were successfully amplified with primer C1-for (
The PrP sequence of gerbils was obtained from GenBank (AF117314).
The GenBank (
We are grateful to Richard Shore (Institute for Terrestrial Ecology, UK) for having donated the breeding pairs of rodents species from which the colonies at ISS originated. We thank Paolo Frassanito and Shimon Simson (Istituto Superiore di Sanità) for animal care, and Consiglia Parisi (Istituto Superiore di Sanità) for administrative management of projects and editorial help.