The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP VRQ -encoding transgenes under an endogenous PrPdeficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP VRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

PilC1, a pilus‐associated protein in Neisseria meningitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact‐regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR‐type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria–cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

The composition, size distribution, and abundance of floating plastic debris in surface waters of the Mediterranean Sea were analyzed in relation to distance to land. We combined data from previously published reports with an intensive sampling in inshore waters of the Northwestern Mediterranean. The highest plastic concentrations were found in regions distant from from land as well as in the first kilometer adjacent to the coastline. In this nearshore water strip, plastic concentrations were significantly correlated with the nearness to a coastal human population, with local areas close to large human settlements showing hundreds of thousands of plastic pieces per km2. The ratio of plastic to plankton abundance reached particularly high values for the coastal surface waters. Polyethylene, polypropylene and polyamides were the predominant plastic polymers at all distances from coast (86 to 97% of total items), although the diversity of polymers was higher in the 1-km coastal water strip due to a higher frequency of polystyrene or polyacrylic fibers. The plastic size distributions showed a gradual increase in abundance toward small sizes indicating an efficient removal of small plastics from the surface. Nevertheless, the relative abundance of small fragments (< 2 mm) was higher within the 1-km coastal water strip, suggesting a rapid fragmentation down along the shoreline, likely related with the washing ashore on the beaches. This study constitutes a first attempt to determine the impact of plastic debris in areas closest to Mediterranean coast. The presence of a high concentration of plastic including tiny plastic items could have significant environmental, health and economic impacts.

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala2(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, The birnaviruses are a family of small icosahedral viruses infecting insects, fish, and birds (15). Only five proteins, generally referred to as VP1, VP2, VP3, VP4, and VP5, are encoded by the viral genome. The Tϭ13 icosahedral birnavirus capsids are made by the VP2 and VP3 proteins. They contain the two double-stranded RNA genomic segments (A and B) and the VP1 protein, a putative RNA-dependent RNA polymerase. Translation of genomic segment A yields a polyprotein, pVP2-VP4-VP3, and a small protein, VP5, of unknown function. The B segment encodes VP1. The polyprotein processing gives rise to VP4, the viral protease, and VP2 and VP3. VP2 carries all the neutralizing epitopes, suggesting that it is at least partly exposed at the outer surface of the capsid. VP3, which interacts with VP1 (16, 24), is thought to be located on the inner surface of the capsid (6). VP3 contains charged residues at its carboxy-terminal domain, a domain suggested to be involved in the genomic RNA interaction. As found for other virus families, the capsid assembly seems to be regulated by polyprotein processing.The infectious bursal disease virus (IBDV), an avian birnavirus, is of major importance to the poultry industry. It causes an immunosuppressive disease in young chickens. After infection, IBDV multiplies rapidly in the B lymphocytes of the bursa of Fabricius, leading to increased susceptibility to other diseases. Very virulent strains have resulted in high rates of mortality in many countries.The first step governing the IBDV capsid assembly is the autoproteolytic cleavage of the polyprotein (1,012 amino acids). This process generates pVP2, VP4, and VP3. The pVP2-to-VP2 conversion involves several proteolytic cleavages at the carboxy end of pVP2 (1,14,20). Based on mutagenesis studies, the putative cleavage site was proposed as defined by the (Thr/ Ala)-X-Ala2Ala motif; three potential sites are present in the C-terminal domain of pVP2 (14).In the present study, we further analyzed the maturation process of VP2. By using mass spectrometry and N-terminal sequence analysis, we s...

Prion diseases are fatal neurodegenerative disorders of animals and humans that are characterized by the conversion of the host-encoded prion protein (PrP) to an abnormal isoform. In several species, including humans, polymorphisms in the gene encoding the PrP protein tightly control susceptibility of individuals toward this disease. In the present study we show that Rov cells expressing an ovine PrP allele ( VRQ PrP) associated with high susceptibility of sheep to scrapie were very sensitive to sheep prion transmission and replicated the agent to high titers. In contrast, we did not find any evidence of infection when Rov cells expressed similar levels of a PrP variant ( ARR PrP) linked to resistance. Our data provide the first direct evidence that natural PrP polymorphisms may affect prion susceptibility by controlling prion replication at the cell level. The study of how PrP polymorphisms influence the genetic control of prion propagation in cultured Rov cells may help elucidate basic mechanisms of prion replication.Transmissible spongiform encephalopathies (TSEs) are fatal degenerative disorders of the central nervous system, which naturally affect animals and humans (for a review, see reference 8). TSEs are associated with the posttranslational conversion of the host-encoded prion protein (PrP) to a conformationally altered form (PrPsc). The causative infectious agents, or prions, are thought to be PrPsc itself or a precursor of it (for reviews, see references 22 and 30).In several species, including mice, sheep, and humans, susceptibility to prion diseases is tightly controlled by the host. The major genetic determinants controlling the length of the incubation period are polymorphisms of Prnp, the PrP-encoding gene. In humans, homozygosity for methionine at the polymorphic residue 129 is associated with high susceptibility to the new variant form of Creutzfeldt-Jakob disease (14), whereas homozygosity for valine is overrepresented among early cases of iatrogenic Creutzfeldt-Jakob disease in the United Kingdom (7). In sheep, variations at three amino acid positions of PrP are predominantly linked to scrapie susceptibility (for a review, see reference 17). The 136 V 154 R 171 Q allele (V, R, and Q stand for valine, arginine, and glutamine, respectively) is associated with extremely high susceptibility to scrapie: in naturally infected flocks, sheep homozygous for VRQ PrP are almost always affected with scrapie. In contrast, the 136 A 154 R 171 R variant (A for alanine) is associated with resistance to the clinical disease. No clinical scrapie case has been reported in hundreds of sheep homozygous for ARR PrP from naturally infected flocks in Europe and the United States, and a single case was reported in Japan (12). Experimental challenge of VRQ-and ARR-encoding sheep with ovine and bovine prions further substantiated the dramatic opposite effects of these two alleles on disease susceptibility, since sheep homozygous for ARR PrP could not be infected by either agent (11). Although the link between specific ...

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Rivers are often challenged by fecal contaminations. The barrier effect of sediments against fecal bacteria was investigated through the use of a microbial source tracking (MST) toolbox, and by Next Generation Sequencing (NGS) of V5-V6 16S rRNA gene (rrs) sequences. Non-metric multi-dimensional scaling analysis of V5-V6 16S rRNA gene sequences differentiated bacteriomes according to their compartment of origin i.e., surface water against benthic and hyporheic sediments. Classification of these reads showed the most prevalent operating taxonomic units (OTU) to be allocated to Flavobacterium and Aquabacterium. Relative numbers of Gaiella, Haliangium, and Thermoleophilum OTU matched the observed differentiation of bacteriomes according to river compartments. OTU patterns were found impacted by combined sewer overflows (CSO) through an observed increase in diversity from the sewer to the hyporheic sediments. These changes appeared driven by direct transfers of bacterial contaminants from wastewaters but also by organic inputs favoring previously undetectable bacterial groups among sediments. These NGS datasets appeared more sensitive at tracking community changes than MST markers. The human-specific MST marker HF183 was strictly detected among CSO-impacted surface waters and not river bed sediments. The ruminant-specific DNA marker was more broadly distributed but intense bovine pollution was required to detect transfers from surface water to benthic and hyporheic sediments. Some OTU showed distribution patterns in line with these MST datasets such as those allocated to the Aeromonas, Acinetobacter, and Pseudomonas. Fecal indicators (Escherichia coli and total thermotolerant coliforms) were detected all over the river course but their concentrations were not correlated with MST ones. Overall, MST and NGS datasets suggested a poor colonization of river sediments by bovine and sewer bacterial contaminants. No environmental outbreak of these bacterial contaminants was detected.