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International audience

International audience; Much remains to be learned about the physical relationship between infectivity and aggregated PrP, and to which extent it varies according to the prion strain. Transmission of natural sheep scrapie and BSE infectious sources to transgenic mice overexpressing the VRQ allele of ovine PrP (tg338 line) has allowed us to establish a panel of biologically cloned strains that have been shown to produce stable and clearly distinct phenotypes based on the electrophoretic profile of PrPSc, its regional distribution in the brain, and the incubation time. We have sought to further compare these strains on the basis of their physicochemical features. We first studied the individual conformational stability of four strains by measuring the resistance to denaturation by guanidine-HCl (GdnHCl). At variance with that recently reported for rodent-adapted strains, we failed to observe a straight correlation between [GdnHCl]1/2 values and incubation times. We next compared the sedimentation properties of abnormal PrP and infectivity of each strain by velocity gradient centrifugation, by using detergent-solubilised, infected brain homogenate instead of PrPres-enriched material. Gradient fractions were analysed for PrPC, PrPSc and PrPres content by ELISA and western blotting, and for infectivity by bioassay in tg338 mice. The detergent treatment conditions were optimised so as to separate most of the PrPC from the bulk of PrPres. PrPres was found to sediment in the middle region of the gradient, however the position and shape of the peak noticeably differed among the strains, reflecting PrPSc aggregates of different sizes. Examining the distribution of infectivity revealed even more striking differences. Thus, 99% of the infectivity was separated from about 90% of PrPres in the case of a fast strain, whereas in the case of BSEov agent the peaks of PrPres and infectivity were largely overlapping. From these results it was concluded that the relationship between infectivity and misfolded PrP multimers is not univocal, suggesting that the size of the most infectious particles may substantially differ among prions strains.

International audience; Much remains unknown about the evolution of the variant Creutzfeldt-Jakob disease (vCJD) epidemic due to uncertainty in ascertaining the prevalence of infection and because the disease might remain asymptomatic or produce an alternate, sporadiclike phenotype. To further model human-to-human vCJD prions spreading, we used a newly developed transgenic mouse model that overexpress human prion protein with methionine at codon 129, the only allele found so far in clinically vCJD-affected patients. These mice were infected with prions derived from variant and sporadic CJD cases by intracerebral or intraperitoneal routes, and were analysed for transmission efficiency and strain phenotype in brain and spleen tissues. After intracerebral challenge, all infected mice developed a typical clinical disease at around 150 days and 500 days for sCJD and vCJD primary isolates, respectively. Of note, one out of four inoculated vCJD cases consistently led to the propagation of both sporadic-like and variant-like CJD prions in the brain, while vCJD-type prions invariably accumulated in the spleen. After intraperitoneal challenge, an inefficient neuroinvasion was observed, resulting in a silent infection with life-long persistence of vCJD prion in the spleen. Our findings have implications in terms of vCJD diagnosis and control. They highlight i) the potential of this agent to establish a subclinical infection in lymphoreticular tissues, thus increasing the level of concern about the risk for iatrogenic transmission; ii) the possibility that person-to-person transmission of vCJD might produce alternative disease phenotypes and that different prions might propagate in different tissues of the same individual.

International audience; Background: In Great Britain, sheep scrapie cases have been detected through active and passive surveillance programmes which do not conform to the characteristics usually associated with classical scrapie. These cases, designated as ‘atypical scrapie’, demonstrate a weak positive PrPres reaction in Bio-Rad ELISA and in histopathology produce a weak immunohistochemistry signal and demonstrate a different PrPSc distribution pattern compared to classical scrapie. Other features, such as an absence of vacuolation at the obex and a characteristic PrPres Western blotting profile are also apparent. Aim: The aim of this Defra-funded study was to determine the transmissibility of atypical scrapie cases from Britain to transgenic PrP and conventional mice, and to examine the biochemical and histopathological characteristics of the transmitted agent(s). Methods: Atypical scrapie isolates were selected from sheep carrying a range of PrP genotypes and inoculated into tg338, C57BL/6 and VM mice. Inoculated mice were clinically monitored and at termination brain samples were examined by Western blotting, lesion profiling and immunohistochemistry. Results: Eighteen atypical scrapie isolates derived from sheep of different genotypes transmitted successfully to tg338 mice at VLA and INRA. Incubation periods, lesion profiles, PrPSc distribution and Western blot profiles of the examined cases were similar, suggesting the isolation of a single strain. Furthermore, the biochemical and histopathological characteristics produced were indistinguishable from those of Nor 98 in the same transgenic mouse line. Bioassay in wild-type mice is ongoing with no TSE positive mice identified up to 431 days post-inoculation. Conclusion: Our findings suggest that the atypical scrapie cases from GB represent infection resulting from a single strain of TSE agent that is indistinguishable from Nor98.

International audience; The ability to infect lymphoreticular organs is a long recognised feature of TSE agents, and there is evidence that extraneural replication can play a pivotal role in the pathogenesis of the infection. We have studied the lymphotropism of sheep scrapie, BSE and CJD infectious sources in transgenic mice expressing either ovineVRQ (tg338) or humanM129 prion protein (tg650) by examining the accumulation of PrPSc and infectivity in their spleen. It was found that tg338 mice intracerebrally inoculated with a class of scrapie primary isolates common in the field accumulated prions with clearly distinct strain properties in the spleen and brain tissues, based on their PrPres molecular profile and biological properties on further passaging. Such a dual distribution was altered upon inoculation by peripheral route. Biological cloning allowed the individualisation of two strain components with fairly different tropisms. One of which, designated Lan19K, appeared to be essentially devoid of lymphotropism, similar to that found for Nor98 atypical scrapie propagated on the same mice. In contrast, another group of scrapie isolates promoted PrPres accumulation at equivalent levels in the spleen and in the brain. These data indicate that the lymphoinvasive potential of natural scrapie strains is quite variable, and that this criterion could be useful as an aid to further investigate their diversity in mouse bioassay. Similar strain-related discrepancies were observed following transmission of classical and atypical BSE agents. Transmission of human TSE isolates to tg650 mice revealed a prominent infection of lymphoid tissue by vCJD agent while only minimal involvement was observed in the case of sCJD agent, thus faithfully reproducing the situation reported in infected humans. Intracerebral inoculation of a reference vCJD sample led to the propagation of prions with either vCJD or sCJD strain-like properties in the brain, but invariably vCJD-like in the spleen. Remarkably, following inoculation by peripheral route, vCJD agent failed to produce an efficient neuroinvasion leading to a clinically silent, sustained extraneural infection along the whole mouse life. Collectively these observations show that prions with different strain properties can propagate concomitantly in the nervous and lymphoid tissues, and point to further complexity in the interactions that natural prion agents can establish with the infected host.

International audience; While multiple conformations of the abnormal prion protein (PrPSc) are thought to encipher prion strain diversity, how they feature in the variable incubation periods observed upon experimental transmission into mice is currently unknown. In order to investigate the natural diversity of sheep scrapie agent, our laboratory has developed a new experimental model consisting of transgenic mice overexpressing the VRQ allele of sheep prion protein (tg338 line). Among 6 potential groups of natural scrapie strains, we have presently stabilised 4 strains that have been biologically cloned and shown to produce stable and distinct phenotypes, based on the incubation time (from 60 to ∼200 days), the molecular profile of PrPSc and its regional distribution in the brain. Moreover these scrapie strains differ in their capacity to replicate in peripheral tissues, such as spleen. For each of these strains, we have examined i) the kinetics of PrPSc accumulation in both brain and spleen, ii) the sensitivity of PrPSc to degradation by either digesting the protein with proteinase K or pepsin in an vitro assay or in vivo by exposing brain homogenates containing PrPP Sc to primary cultures of macrophages, iii) the resistance to denaturation with increasing concentration of guanidine-HCl. These data will be presented and their possible significance in term of strain biology will be discussed.

International audience; In conventional mice, murine strains of prions show highly specific phenotypes, as defined by several parameters including incubation period of disease, nature of clinical signs, western blot profile of abnormal prion protein (PrPSc), distribution of vacuoles and PrPSc in the brain. More surprisingly, this remains independent of where prions have had access to the brain, supporting the view that, through a determinism which remains obscure, the multiplication of the infectious agent and the PrP conversion process is favoured in brain specific areas. We sought to examine if the inoculation of natural scrapie strains to transgenic mice expressing ovine PrP would lead to similar findings. We have therefore 1) analysed the phenotype of the cloned ovine prion strain 127S after intraperitoneal or intracerebral infection of tg338 mice overexpressing the VRQ allele of sheep prion protein; 2) compared the cerebral PrPSc distribution of this strain with another cloned and phenotypically distinct one, LAN19K, after intravitreal injection. Our results show that 1) the apparent phenotype of the 127S strain is clearly influenced by the route of infection since intraperitoneally versus intracerebrally tg338 mice show different clinical signs, levels of PrPSc accumulation and distribution pattern of PrPSc and vacuoles in brain; 2) 127S and LAN19K show a similar propagation into the brain until the terminal stage of the disease, which was mainly restricted to the ocular projections. This common PrPSc distribution pattern strikingly differed from that obtained after intracerebral or intraperitoneal inoculation of these strains. Our data therefore suggest that, depending on the model, the capacity of prion strains to replicate in brain specific areas may be influenced by the route of infection.

National audience

National audience; Serial transmission to a panel of congenic mouse lines, where characteristics of incubation time and brain vacuolation areas give distinct patterns, is the current way of investigating strain biological diversity in natural sheep scrapie. Following inoculation of natural scrapie isolates to mice, the disease commonly requires 1 or 2 years to develop, and a proportion of isolates fail to be transmitted. As a consequence, a limited number of isolates have been studied yet, and the extent of the natural scrapie strain variation remains largely unexplored. In an effort to develop an improved model for transmission of sheep scrapie, we have expanded a series of transgenic mice (tgOv) that express the VRQ (Val136-Arg154-Gln171) allele of ovine PrP on a null mouse PrP background. This allele is associated with the highest susceptibility to the disease in many flocks. Mice of the tgOv line tg338, expressing high PrP level (~ 10-fold that in sheep brain), were inoculated intracerebrally with more than 40 scrapie sources including sheep of distinct PrP genotypes and of various geographical areas in Europe. In contrast with earlier studies using non-transgenic mice, all isolates transmitted successfully, generally with a ~ 100% attack rate. However, the incubation time to death differed widely among the isolates, ranging from 70 to 750 days. More than half of the isolates transmitted in

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National audience

National audience