Stefano Marcon

Istituto Superiore di Sanità, Roma, Latium, Italy

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Publications (12)26.04 Total impact

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    ABSTRACT: 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.
    07/2013; 23390:446-456.
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    ABSTRACT: 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.
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    ABSTRACT: Procedures for discriminating scrapie from bovine spongiform encephalopathy (BSE) in sheep are relevant to ascertain whether BSE has entered the sheep population. This study was aimed at investigating whether the suitability of an official EU discriminative method is affected by the sheep PrP genotype and route of infection.
    Journal of General Virology 02/2012; 93(Pt 2):450-5. · 3.13 Impact Factor
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    ABSTRACT: Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2) values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2) values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.
    PLoS ONE 01/2010; 5(9):e12723. · 3.53 Impact Factor
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    ABSTRACT: Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.
    PLoS One 5 (9) (2010). 01/2010;
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    ABSTRACT: The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.
    Journal of Virology 08/2007; 81(13):7306-9. · 5.08 Impact Factor
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    ABSTRACT: The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.
    Journal of Virology 06/2007; 81(9):4872-6. · 5.08 Impact Factor
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    ABSTRACT: The association between PrP gene variations and scrapie susceptibility was studied in a single herd of Ionica breed goats. The entire herd comprised 100 animals, 11 of which were clinically affected and showed pathological prion protein (PrP(Sc)) deposition in both their central nervous system (CNS) and lymphoreticular system (LRS). Among asymptomatic goats, nine harboured PrP(Sc) in both CNS and LRS, 19 showed PrP(Sc) only at the LRS level and 61 animals had no PrP(Sc) deposition. Genetic analysis of the PrP gene coding sequence revealed the presence of several polymorphisms, namely G37V, T110P, H143R, R154H, Q222K and P240S. Silent polymorphisms were also found at codons 42, 138, 219 and 232. The effect of PrP polymorphism on scrapie susceptibility was assessed by comparing the genotype distribution at each locus among animals with different pathogenetic and clinical disease stages. Significant differences in the distribution of genotypes were observed for codons 154 and 222, with polymorphism at codon 154 modulating susceptibility to scrapie and lysine at codon 222 being associated with scrapie resistance. The allelic variant encoding lysine at position 222 could be a valuable candidate to select in the framework of appropriate breeding programmes for scrapie resistance in goats.
    Journal of General Virology 06/2006; 87(Pt 5):1395-402. · 3.13 Impact Factor
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    ABSTRACT: The application of a selective culling programme in two scrapie affected flocks of Massese breed sheep is described. The genetic susceptibility of this breed and the sensitivity of different diagnostic methods in the pre-clinical diagnosis of scrapie were also investigated. Overall, 2,068 clinically healthy sheep underwent PrP genotyping, providing the basis for selective culling. The prevalence of scrapie infection was investigated in susceptible sheep by two independent diagnostic methods. All the sheep older than 18 months (n = 620) were tested by Prionics Check Western rapid test on the obex, with a prevalence of infection of 3.9%. Furthermore, 385 sheep underwent immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN), with a prevalence of infection of 5.2%. Overall, 32 sheep were diagnosed with pre-clinical scrapie. Of these, 31 were positive by Western blot on the spleen, 29 by IHC on the RPLN and tonsil, 28 by IHC on the obex, 24 by rapid test, and only 18 by IHC on the third eyelid. All the scrapie positive sheep were of the ARQ/ARQ, ARQ/AHQ or ARQ/VRQ genotypes. No significant differences in scrapie prevalence were observed among these genotypes. The estimated risk of the three targeted alleles was also similar, suggesting that in this breed the VRQ allele was not at higher risk for scrapie, compared to the ARQ and AHQ alleles.
    Archives of Virology 11/2005; 150(10):1959-76. · 2.03 Impact Factor
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    ABSTRACT: Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP(Sc) from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP(Sc). Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP(Sc) from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP(Sc). Scrapie cases displayed homogeneous PrP(Sc) features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP(Sc). While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.
    Journal of Clinical Microbiology 10/2003; 41(9):4127-33. · 4.07 Impact Factor
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    ABSTRACT: Gli autori descrivono i quadri istopatologici e l’immunolocalizzazione della GFAP e della PrP<sub>sc</sub> in 25 aree del SNC di 8 ovini con scrapie naturale, provenienti da due allevamenti della Sardegna. Il quadro clinico e lesivo sostanzialmente omogeneo portano a supporre una identità del ceppo, o dei ceppi conoivoli nei due focolai di malattia.

Publication Stats

184 Citations
26.04 Total Impact Points

Institutions

  • 2005–2012
    • Istituto Superiore di Sanità
      • Department of Food Safety and Veterinary Public Health
      Roma, Latium, Italy
  • 2007
    • Istituto Zooprofilattico Sperimentale delle Venezie
      Padua, Veneto, Italy
  • 2003–2007
    • L'Istituto Zooprofilattico Sperimentale del Lazio e Toscana
      Roma, Latium, Italy