Juan-María Torres

Early detection of PrPres in BSE-infected bovine PrP transgenic mice.

Summary. Transgenic mouse lines expressing different levels of the bovine prion protein gene (boPrPC) were generated. Upon infection with BSE prions, all transgenic lines tested exhibited characteristics of the bovine disease. Typical CNS spongiform degeneration was observed by histopathology and presence of PrPres could be detected both by Western blot and immunohistochemistry (IHC) assays, confirming for this model the absence of an interspecies barrier to BSE infection. Differences in incubation times post-inoculation depend upon the expression level of boPrPC and the amount of prions in the inoculum. In the absence of clinical signs, pathognomonic markers of disease could be detected as early as 150 or 196 days post-inoculation by IHC and Western blot analysis, respectively. This result indicates that prion infectivity in experimental mouse bioassays can be measured earlier by assessing immunologically the presence of PrPres in brains from inoculated animals. Although these transgenic mice were also susceptible to sheep scrapie prion infection, the extent of incubation times was considerably longer and PrPres was detected in only 70 % of inoculated mice. Interestingly, transgenic mice-propagated sheep scrapie prions displayed distinct biochemical properties when compared to both the original sheep scrapie and transgenic mouse-propagated BSE inoculum.

Evidence for zoonotic potential of ovine scrapie prions

Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.

Transgenic Mice Expressing Porcine Prion Protein Resistant to Classical Scrapie but Susceptible to Sheep Bovine Spongiform Encephalopathy and Atypical Scrapie

Atypical scrapie strain phenotypes may shift when transmitted to a new host.

Emergence of Classical BSE Strain Properties during Serial Passages of H-BSE in Wild-Type Mice

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 (PrPd) 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, PrPd 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.

Different Behavior toward Bovine Spongiform Encephalopathy Infection of Bovine Prion Protein Transgenic Mice with One Extra Repeat Octapeptide Insert Mutation

In humans, insert mutations within the repetitive octapeptide region of the prion protein gene (Prnp) are often associated with familial spongiform encephalopathies. In this study, transgenic mice expressing bovine PrP (boTg mice) bearing an additional octapeptide insertion to the wild type (seven octapeptide repeats instead of six) showed an altered course of bovine spongiform encephalopathy (BSE) infection, reflected as reduced incubation times when compared with boTg mice expressing similar levels of the wild-type six-octapeptide protein. In both boTg mouse lines (bo6ORTg and bo7ORTg), incubation times were affected drastically depending on transgene expression levels and the inoculum used. In accordance with the lack of an interspecies barrier to BSE infection, we detected the typical signs of CNS spongiform degeneration by histopathological analysis and the presence of the bovine prion PrPres by Western blot or immunohistochemical analyses. When 7OR-PrPres was propagated in bo7ORTg mice, a similar earlier onset of clinical signs was observed compared with bo6ORTg mice. Proteins PrPC and PrPres containing seven octapeptides (7OR-PrPC and 7OR-PrPres) showed similar protease sensitivity and insolubility in nondenaturing detergents to homologous 6OR-PrPC and 6OR-PrPres. In addition, bo7ORTg mice showed a higher sensitivity than bo6ORTg mice for detecting prion infection in specimens previously diagnosed as negative by conventional biochemical techniques. In the absence of clinical signs of disease, 7OR-PrPres could be detected as early as 120 d after inoculation by immunohistochemical and Western blot analyses. These findings may help us improve the current mouse bioassays and understand the role of the octapeptide repeat region in susceptibility to disease.

Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

TOC Summary: An epidemic agent could have originated from such a cattle prion.

Vertical Transmission of Bovine Spongiform Encephalopathy Prions Evaluated in a Transgenic Mouse Model

ABSTRACT In this work we show evidence of mother-to-offspring transmission in a transgenic mouse line expressing bovine PrP (boTg) experimentally infected by intracerebral administration of bovine spongiform encephalopathy (BSE) prions. PrPres was detected in brains of newborns from infected mothers only when mating was allowed near to the clinical stage of disease, when brain PrPres deposition could be detected by Western blot analysis. Attempts to detect infectivity in milk after intracerebral inoculation in boTg mice were unsuccessful, suggesting the involvement of other tissues as carriers of prion dissemination. The results shown here prove the ability of BSE prions to spread centrifugally from the central nervous system to peripheral tissues and to offspring in a mouse model. Also, these results may complement previous epidemiological data supporting the occurrence of vertical BSE transmission in cattle.

Transgenic mice expressing bovine PrP with a four extra repeat octapeptide insert mutation show a spontaneous, non‐transmissible, neurodegenerative disease and an expedited course of BSE infection

Transgenic (Tg) mice carrying four extra octapeptide repeats (OR) in the bovine PrP gene (10OR instead of 6) have been generated. In these mice, neuropathological changes were observed depending upon the level of transgene expression. These changes primarily involved a slowly advancing neurological disorder, characterized clinically by ataxia, and neuropathologically, by vacuolization in different brain areas, gliosis, and loss of cerebellar granule cells. Accumulation of insoluble bovine 10OR‐PrP (bo10OR‐PrP) was observed depending on the level of expression but no infectivity was found associated with this insoluble form. We also compared the behavior of bo6OR‐PrP and bo10OR‐PrP Tg mouse lines in response to BSE infection. BSE‐inoculated bo10ORTg mice showed an altered course of BSE infection, reflected by reduced incubation times when compared to bo6ORTg mice expressing similar levels of the wild type 6OR‐PrP. In BSE‐inoculated mice, it was possible to detect PrPres in 100% of the animals. While insoluble bo10OR‐PrP from non‐inoculated bo10ORTg mice was non‐infectious, brain homogenates from BSE‐inoculated bo10ORTg mice were highly infectious in all the Tg mouse lines tested. This Tg mouse model constitutes a new way of understanding the pathobiology of bovine transmissible spongiform encephalopathy. Its potential applications include the assessment of new therapies against prion diseases.

Isolation of an attenuated myxoma virus field strain that can confer protection against myxomatosis on contacts of vaccinates.

Summary. Twenty MV strains obtained from a survey of field strains currently circulating throughout Spain were analyzed for their virulence and horizontal spreading among rabbits by contact transmission. A virus strain with suitable characteristics to be used as a potential vaccine against myxomatosis in wild rabbit populations was selected. Following inoculation, the selected MV strain elicited high levels of MV specificantibodies and induced protection of rabbits against a virulent MV challenge. Furthermore, the attenuated MV was transmitted to 9 out of 16 uninoculated rabbits by contact, inducing protection against myxomatosis.

Elements Modulating the Prion Species Barrier and Its Passage Consequences

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor. As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.