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Yellow Stripe-Like (YSL) proteins are a family of plant transporters typically involved in transition metal homeostasis. The substrate of three of the four YSL clades (clades I, II, and IV) are metal complexes with non-proteinogenic amino acid nicotianamine or its derivatives. No such transport capabilities have been shown for any member of the remaining clade (clade III), which is able to translocate short peptides across the membranes instead. The connection between clade III YSL members and metal homeostasis might have been masked by the functional redundancy characteristic of this family. This might have been circumvented in legumes through neofunctionalization of YSLs to ensure a steady supply of transition metals for symbiotic nitrogen fixation in root nodules. To test this possibility,Medicago truncatulaclade III transporter MtYSL7 has been studied both when the plant was fertilized with ammonium nitrate or when nitrogen had to be provided by endosymbiotic rhizobia within the root nodules. MtYSL7 is a plasma membrane protein expressed in the vasculature and in the nodule cortex. This protein is able to transport short peptides into the cytosol, although none with known metal homeostasis roles. ReducingMtYSL7expression levels resulted in diminished nitrogen fixation rates. In addition, nodules of mutant lines lackingYSL7accumulated more copper and iron, the later the likely result of increased expression in roots of iron uptake and delivery genes. The available data is indicative of a role of MtYSL7, and likely other clade III YSLs, in transition metal homeostasis.ONE SENTENCE SUMMARYMedicago truncatulaYSL7 is a peptide transporter required for symbiotic nitrogen fixation in legume nodules, likely controlling transition metal allocation to these organs.

27 Symbiotic nitrogen fixation in legume root nodules requires a steady supply of 28 molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient 29 has to be provided by the host plant from the soil, crossing several symplastically 30 disconnected compartments through molybdate transporters, including members of the 31 MOT1 family. MtMOT1.2 is a Medicago truncatula MOT1 family member located in 32 the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 33 indicates that it is associated to the plasma membrane and to intracellular membrane 34 systems, where it would be transporting molybdate towards the cytosol, as indicated in 35 yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth 36 compared to wild-type plants when nitrogen fixation was required, but not when nitrogen 37 was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase 38 activity was observed, nitrogenase activity was severely affected, explaining the observed 39 difference of growth depending on nitrogen source. This phenotype was the result of 40 molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with 41 a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both 42 restored wild-type levels of growth and nitrogenase activity. These results support a 43 model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into 44 the nodules. 45 46, 19 molybdate delivery for nitrogen fixation is occurring at the 450 level of nodule vessels and 451 not in loading the root vasculature with Mo. Otherwise, an accumulation of Mo in mot1.2 452 roots would be expected as well as a decrease in shoots, and none was detected in either 453 (in this case, even slightly higher levels were detected, which might correspond to surplus 454 Mo being delivered to the shoots). 455 In summary, MtMOT1.2 would position itself between molybdate root uptake 456 transporter, likely MtMOT1.1 as the closest LjMOT1 orthologue, and the nodule apoplast 457 molybdate uptake protein MtMOT1.3 (Fig. 6). MtMOT1.2 would facilitate the transfer 458 of this nutrient into endodermal cells mediating the sink-to-source molybdate trafficking, 459 which would be controlled by mass-effects to ensure that it reaches its destination. 460 However, a critical point remains to be solved, which is the identity of the proteins 461 mediating molybdate efflux from the cytosol to the symbiosome. Whether these are 462 sulfate transporters, or whether a novel family of Mo transporters with a direction of 463 transport opposite to MOT1 proteins, remains to be unveiled. 464 465 ACKNOWLEDGEMENTS 466 This research was funded by a European Research Council Starting Grant (ERC- 467 2013-StG-335284) and a Spanish Ministry of Economy and Competitiveness grant 468 (AGL2015-65866-P) to M.G-G. Development of the M. truncatula Tnt1 mutant 469 population was, in part, funded by the National Science Foundation, USA (DBI-0703285 470 & IOS-1127155) to K.S.M. Yeast transport assays were partially funded by the Plan 471 Propio de la Universidad de Córdoba (to M.T-J) and MINECO (BFU2015-70649-P). The 472 authors would like to thank Dr. Emilio Fernández and Dr. Aurora Galván (Universidad 473 de Córdoba) for their help with the yeast transport assays, as well as to members of 20 Laboratory 281 at Centro de Biotecnología y Genómica de 474 Plantas (UPM-INIA) for their 475 support and feed-back in preparing this manuscript

International audience; Eilat virus (EILV) is described as one of the few alphaviruses restricted to insects. We report the record of a nearly-complete sequence of an alphavirus genome showing 95% identity with EILV during a metagenomic analysis performed on 1488 unblood-fed females and 1076 larvae of the mosquito Culex pipiens captured in Rabat (Morocco). Genetic distance and phylogenetic analyses placed the EILV-Morocco as a variant of EILV. The observed infection rates in both larvae and adults suggested an active circulation of the virus in Rabat and its maintenance in the environment either through vertical transmission or through horizontal infection of larvae in breeding sites. This is the first report of EILV out of Israel and in Culex pipiens populations.

SummaryMolybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters.Members of the molybdate transporters family MOT1 were identified in the model legumeMedicago truncatulaand their expression in nodules determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of aTnt1insertional mutant line were carried out in the oneM. truncatulaMOT1 family member expressed specifically in nodules.Among the five MOT1 members present inM. truncatulagenome,MtMOT1.3is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. Amot1.3-1knockout mutant showed an impaired growth concomitant with a reduction in nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore,mot1.3-1plants transformed with a functional copy ofMtMOT1.3showed a wild type-like phenotype.These data are consistent with a model in which MtMOT1.3 would be responsible for introducing molybdate into nodule cells, which will be later used to synthesize functional nitrogenase.

Background Avian influenza viruses (AIV) have been detected in wild birds in West Africa during the northern winter, but no information is available on a potential year-round circulation of AIV in West Africa. Such year-round circulation would allow reassortment opportunities between strains circulating in Afrotropical birds and strains imported by migratory birds wintering in West Africa. Objective and Method A 2-year longitudinal survey was conducted in the largest continental wetland of West Africa, the Inner Niger Delta in Mali, to determine the year-round circulation of AIV in wild birds. Results and Conclusions Avian influenza virus RNA was detected during all periods of the year. Very low prevalence was detected during the absence of the migratory wild birds. However, a year-round circulation of AIV seems possible in West Africa, as shown in other African regions. West Africa may hence be another potential site of reassortment between AIV strains originating from both Afro-tropical and Eurasian regions. (Resume d'auteur)

International audience; To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.

This study reports the first evidence of circulation of avian influenza viruses (AIV) in domestic poultry in Mali. In the Mopti region, where AIV have already been isolated in migratory water birds, we sampled 223 backyard domestic birds potentially in contact with wild birds and found that 3.6% had tracheal or cloacal swabs positive by real-time reverse transcription PCR (rRT-PCR) for type A influenza viruses (IVA) and that 13.7% had sera positive by commercial ELISA test detecting antibodies against IVA. None of the birds positive by rRT-PCR for IVA was positive by rRT-PCR for H5 and H7 subtypes, and none showed any clinical signs therefore indicating the circulation of low pathogenic avian influenza. Unfortunately, no virus isolation was possible. Further studies are needed to assess the temporal evolution of AIV circulation in the Mopti region and its possible correlation with the presence of wild birds.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza., Author Summary Influenza viruses, including H5N1 bird influenza viruses continue to form a major threat for public health. Available antiviral drugs for the treatment of influenza are effective to a limited extent and the emergence of resistant viruses may further undermine their use. The symptoms associated with influenza are caused by replication of the virus in the respiratory tract and the host immune response. Here, we report that a molecule of the fibrinolytic system, plasminogen, contributes to inflammation caused by influenza. Inhibiting the action of plasminogen protected mice from severe influenza infections, including those caused by H5N1 and H1N1 pandemic 2009 viruses and may be a promising novel strategy to treat influenza.

International audience; Newcastle disease (ND) is one of the most lethal diseases of poultry worldwide. It is caused by an avian paramyxovirus 1 that has high genomic diversity. In the framework of an international surveillance program launched in 2007, several thousand samples from domestic and wild birds in Africa were collected and analyzed. ND viruses (NDV) were detected and isolated in apparently healthy fowls and wild birds. However, two thirds of the isolates collected in this study were classified as virulent strains of NDV based on the molecular analysis of the fusion protein and experimental in vivo challenges with two representative isolates. Phylogenetic analysis based on the F and HN genes showed that isolates recovered from poultry in Mali and Ethiopia form new groups, herein proposed as genotypes XIV and sub-genotype VIf with reference to the new nomenclature described by Diel's group. In Madagascar, the circulation of NDV strains of genotype XI, originally reported elsewhere, is also confirmed. Full genome sequencing of five African isolates was generated and an extensive phylogeny reconstruction was carried out based on the nucleotide sequences. The evolutionary distances between groups and the specific amino acid signatures of each cluster allowed us to refine the genotype nomenclature.

International audience; Heterogeneity in the transmission rates of pathogens across hosts or environments may produce disease hotspots, which are defined as specific sites, times or species associations in which the infection rate is consistently elevated. Hotspots for avian influenza virus (AIV) in wild birds are largely unstudied and poorly understood. A striking feature is the existence of a unique but consistent AIV hotspot in shorebirds (Charadriiformes) associated with a single species at a specific location and time (ruddy turnstone Arenaria interpres at Delaware Bay, USA, in May). This unique case, though a valuable reference, limits our capacity to explore and understand the general properties of AIV hotspots in shorebirds. Unfortunately, relatively few shorebirds have been sampled outside Delaware Bay and they belong to only a few shorebird families; there also has been a lack of consistent oropharyngeal sampling as a complement to cloacal sampling. In this study we looked for AIV hotspots associated with other shorebird species and/or with some of the larger congregation sites of shorebirds in the old world. We assembled and analysed a regionally extensive dataset of AIV prevalence from 69 shorebird species sampled in 25 countries across Africa and Western Eurasia. Despite this diverse and extensive coverage we did not detect any new shorebird AIV hotspots. Neither large shorebird congregation sites nor the ruddy turnstone were consistently associated with AIV hotspots. We did, however, find a low but widespread circulation of AIV in shorebirds that contrast with the absence of AIV previously reported in shorebirds in Europe. A very high AIV antibody prevalence coupled to a low infection rate was found in both first-year and adult birds of two migratory sandpiper species, suggesting the potential existence of an AIV hotspot along their migratory flyway that is yet to be discovered.

Despite considerable effort for surveillance of wild birds for avian influenza viruses (AIVs), empirical investigations of ecological drivers of AIV prevalence in wild birds are still scarce. Here we used a continental-scale dataset, collected in tropical wetlands of 15 African countries, to test the relative roles of a range of ecological factors on patterns of AIV prevalence in wildfowl. Seasonal and geographical variations in prevalence were positively related to the local density of the wildfowl community and to the wintering period of Eurasian migratory birds in Africa. The predominant influence of wildfowl density with no influence of climatic conditions suggests, in contrast to temperate regions, a predominant role for inter-individual transmission rather than transmission via long-lived virus persisting in the environment. Higher prevalences were found in Anas species than in non- Anas species even when we account for differences in their foraging behaviour (primarily dabbling or not) or their geographical origin (Eurasian or Afro-tropical), suggesting the existence of intrinsic differences between wildfowl taxonomic groups in receptivity to infection. Birds were found infected as often in oropharyngeal as in cloacal samples, but rarely for both types of sample concurrently, indicating that both respiratory and digestive tracts may be important for AIV replication.

A one-step real-time Taqman® RT-PCR assay (RRT-PCR) for peste des petits ruminants virus (PPRV) was developed to detect the four lineages of PPRV by targeting the nucleoprotein (N) gene of the virus. This new assay was compared to a conventional RT-PCR on reference strains and field materials. Quantitation was performed against a standard based on a synthetic transcript of the NPPR gene for which a minimum of 32 copies per reaction were detected with a corresponding Ct value of 39. Depending on the lineage involved, the detection limit of RRT-PCR was decreased by one to three log copies relative to the conventional method. The lower stringency occurred with lineage III because of minor nucleotide mismatches within the probe region. The assay did not detect phylogenetically or symptomatically related viruses of ruminants (such as rinderpest, bluetongue, and bovine viral diarrhea viruses). However, it was capable of detecting 20% more positive field samples with low viral RNA loads compared to the conventional PCR method. When compared on a proficiency panel to the method developed by Bao et al. (2008), the sensitivity of the in-house assay was slightly improved on lineage II. It proved significantly faster to perform and hence better adapted for monitoring large numbers of at risk or diseased animals. © 2010 Elsevier B.V.

International audience

International audience; The large (L) polymerase gene and the 5'-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl., Author Summary Until recently, the highly pathogenic avian influenza (HPAI) viruses responsible for high mortality in some domestic poultry were considered not to have a wild bird reservoir, but to emerge in domestic poultry populations from low pathogenic viruses perpetuated in wild waterbirds. The rapid spread of H5N1 HPAI virus in 2005–2006, with concurrent outbreaks reported in both domestic and wild birds over Asia, Europe, and Africa, has raised concerns about the potential role of migratory birds in the epidemiology of the HPAI infection. Wild birds were sampled in Africa and tested by molecular and virological methods in an attempt to trace the circulation of HPAI viruses. In addition, some of these wild birds were equipped with satellite transmitters to track their local and migratory movements in relation to the potential spread of avian diseases. Avian influenza viruses (H5N2) were detected in wild waterfowl in Nigeria, and were subsequently characterized as highly pathogenic by molecular sequencing (HPAI viral genotype). Movements of one infected bird tracked by satellite telemetry revealed that it survived infection by an HP viral genotype. This result constitutes a rare finding of infection by an AIV with an HPAI viral genotype in healthy wild birds.