Detection of potential hotspots of AIV infection in shorebirds sampled at various sites across Eurasian and Afro-tropical regions (<b>Figure 1</b>-B; and supporting information Table S2).

<p>The number of AIV-positive birds detected in relation to the number of birds sampled per sampling occasion is here compared to the threshold number of positive birds (solid line) below which the prevalence is unlikely (probability <0.05) to be greater than 10% for a sample of the same size. Points on or above the line represent potential AIV hotspots, i.e. sampling occasions (n = 5) for which the number of positive birds was too large for rejecting the hypothesis that prevalence could be >10% (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone-0046049-t003" target="_blank">Table 3</a>). Only sampling occasions (n = 47) that had at least 28 birds sampled were considered in this analysis.</p

List of potential hotspots of AIV infection detected in our study.

<p>These sites correspond to sampling occasions (top) at which the number of AIV-positive birds was above the threshold number of birds for which the hypothesis that prevalence is lower than 10% could not be rejected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone-0046049-g002" target="_blank">Figure 2</a>). Sampling conducted in different years at the same sites during the same months and on the same species (below) detected a low number of AIV-positive birds.</p>a<p>. African jacana <i>Actophilornis africana</i>, Kittlitz's plover <i>Charadrius pecuarius</i>, Little stint <i>Calidris minuta</i>, Blacksmith lapwing <i>Vanellus armatus</i>, Slender-billed gull <i>Chroicocephalus genei</i>, Curlew sandpiper Calidris ferruginea, Wood sandpiper <i>Tringa glareola</i>.</p

Mean seroprevalence of AIV antibodies among closely related shorebird species in relation to the mean latitude of their breeding range.

<p>Seroprevalence were measured in West Africa (the Banc d'Arguin, Mauritania and the Inner Niger Delta, Mali) and the Delaware Bay, USA (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Stallknecht1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Brown2" target="_blank">[31]</a>) using the same commercial bELISA kit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#s2" target="_blank">Methods</a>). The mean species breeding latitude was computed from the northern and southern limits of the breeding distribution of the populations present at each site using distribution maps from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Jourdain1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Poole1" target="_blank">[32]</a>. Species include ruddy turnstone (Δ), red knot (□), dunlin (o), sanderling (◊), short-billed dowitcher (×), ruff and wood sandpiper (−). Error bars represent the binomial exact 95% confidence interval.</p

Location of the world's largest congregation sites of waders (sandpipers, plovers and allies), i.e. sites where at least 500,000 birds congregate annually.

<p>Among these sites where birds have been tested for AIV infection (black symbols - from the literature; grey symbols - this study) an AIV hotspot has been reported only at the Delaware Bay (no. 7). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049.s007" target="_blank">Table S6</a> (supporting information) for detailed information on each site. 1-Yukon-Kuskokwim Delta, 2-Copper River Delta, 3-Fraser' River Estuary, 4-Gray's harbour estuary, 5-Bay of Fundy, 6-Great Salt Lake, 7-Delaware Bay, 8-Cheyenne Bottoms, 9-San Francisco Bay, 10-Bahia de Santa Maria, 11-Upper Bay of Panama, 12-Suriname coast, 13-Laguna Mar Chiquita, 14-Wadden Sea, 15-Rhine-Maas-Schelde Delta, 16-Azov Sea, 17-Sea of Okhotsk, 18-Tengiz-Korgalzhyn Lakes, 19-Yellow Sea coast, 20-Arabian Sea off Oman, 21-Banc d'Arguin, 22-Senegal River Delta, 23-Bijagos Archipelago (map by M. Gély ©Cirad).</p

Location of the study sites.

<p>(A) The Banc d'Arguin (Mauritania) and the main shorebirds migratory flyways across Western Eurasia and Africa. (B) All shorebird sampling sites considered in our study (list of sites ranked by latitude: Ukraine - Eastern Sivash, Romania-Danube Delta, Turkey - Kizilirmak Delta and Yumurtalik Lagoons, Iran - Fereydoon Kenar marshes, Morocco - Marais du Bas Loukkos and Sidi Moussa-Oualidia Lagoon, Tunisia - Thyna salt pans, Egypt - Nile River Delta and Lake Qarun, Mauritania - Banc d'Arguin National Park, Senegal/Mauritania - Senegal River Delta, Republic of Sudan - El Saggay Island, Mali - Inner Niger Delta, Niger - Kurfunkura pond and Gaya, Chad - Lake Chad, Nigeria - Hadejia-Nguru wetlands, Burkina Faso - Lake Kompienga, Ethiopia - Lake Debre Zeit, South Sudan - Bargel wetland, Kenya - Lakes around Nairobi, Tanzania - Lake Manyara, Malawi - Lake Chilwa, Zambia - Kafue Flats, Zimbabwe - Lakes Manyame-Chivero, Botswana - Lake Ngami, Mozambique - Massingir Dam and Lake Chuali, South Africa - Barberspan wetland and Strandfontein). A detailed list of sampling sites is provided in the supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049.s003" target="_blank">Table S2</a> (map by M. Gély ©Cirad).</p

List of the explanatory variables tested to explain geographical, seasonal and species variations in AIV prevalence in shorebirds across Eurasian and Afro-tropical regions (Figure 1).

a<p>. Only tested for species from the Scolopaci and Charadrii clades.</p

Results of the model selection procedure relating variations in AIV prevalence in shorebirds across Eurasia and Africa to species ecological traits, sampling procedure, period and ecological characteristics of the sampling site.

<p>All models were fitted as generalized mixed effects models, with sampling occasion fitted as random intercept terms to control for pseudo-replication and other explanatory variables as fixed effect. The initial full model was simplified by backwards elimination of non-significant variables. Coefficient estimates are given only for variables interpreted as statistically significant (t-test,* p<0·05) and included in the minimal adequate model (NS - no coefficient of individual species was statistically different from zero). The test statistics refer to a likelihood ratio test between the model in which the variable is retained and in which it is excluded. Statistics for variables not included in the final model correspond to the values when added to the minimal adequate model. Variables related to species traits were included in substitution to the variable species.</p

List of specific signatures on the F complete gene for genotypes and subsequent sub-genotype discrimination.

<p>The second column indicates the combination of specific amino (first line) and nucleic (second line) signatures that discriminate the genotypes. The fourth column shows the same for the sub-genotype discrimination and is applicable once the genotype has been assigned. Underlined amino or nucleic acids represent specific unique signatures.</p

Primers used for complete sequencing of the F and HN genes.

a<p>F, forward; R, reverse.</p>b<p>M, F, HN, and L matrix, fusion protein, hemagglutinin-neuraminidase, and polymerase genes, respectively.</p>c<p>primers designed on conserved sequences based on the alignment of the complete sequences of 219 F genes and 74 HN genes published in GenBank.</p

Characteristics of viruses analyzed in this study.

<p><b>-</b> *full genome sequence.</p