Yb-to-Eu Cooperative Sensitization Upconversion in a Multifunctional Molecular Nonanuclear Lanthanide Cluster in Solution.

Lanthanide metal clusters excel in combining molecular and material chemistry properties. Here, we report an efficient cooperative sensitization UC phenomenon of a Eu3+/Yb3+ nonanuclear lanthanide cluster in CD3OD. The synthesis and characterization of the heteronuclear cluster in the solid state and solution are described together with the UC phenomenon showing Eu3+ luminescence in the visible region upon 980 nm NIR excitation of Yb3+ at concentrations as low as 100 nM. Alongside being the Eu/Yb cluster to display UC (with a quantum yield value of 4.88 × 10-8 upon 1.13 W cm-2 excitation at 980 nm), the cluster exhibits downshifted light emission of Yb3+ in the NIR region upon 578 nm visible excitation of Eu3+, which is ascribed to sensitization pathways for Yb through the 5D0 energy levels of Eu3+. Additionally, a faint emission is also observed at ca. 500 nm upon 980 nm excitation, originating from the cooperative luminescence of Yb3+. The [Eu8Yb(BA)16(OH)10]Cl cluster (BA = benzoylacetonate) is also a field-induced single-molecular magnet (SMM) under 4K with a modest Ueff/kB of 8.48 K, thereby joining the coveted list of Yb-SMMs and emerging as a prototype system for next-generation devices, combining luminescence with single-molecular magnetism in a molecular cluster.

Synthesis and Photophysical Properties of Lanthanide Pyridinylphosphonic Tacn and Pyclen Derivatives: From Mononuclear Complexes to Supramolecular Heteronuclear Assemblies.

Synthetic methodologies were developed to achieve the preparation of ligands L1 and L2 consisting of tacn- and pyclen-based chelators decorated with pyridinylphosphonic pendant arms combined with ethylpicolinamide or acetate coordinating functions, respectively. Phosphonate functions have been selected for their high affinity toward Ln3+ ions compared to their carboxylated counterparts and for their steric hindrance that favors the formation of less-hydrated complexes. Thanks to regiospecific N-functionalization of the macrocyclic backbones, the two ligands were isolated with good yields and implicated in a comprehensive photophysical study for the complexation of Eu3+, Tb3+, and Yb3+. The coordination behavior of L1 and L2 with these cations has been first investigated by means of a combination of UV-vis absorption spectroscopy, steady-state and time-resolved luminescence spectroscopy, and 1H and 31P NMR titration experiments. Structural characterization in solution was assessed by NMR spectroscopy, corroborated by theoretical calculations. Spectroscopic characterization of the Ln3+ complexes of L1 and L2 was done in water and D2O and showed the effective sensitization of the lanthanide metal-centered emission spectra, each exhibiting typical lanthanide emission bands. The results obtained for the phosphonated ligands were compared with those reported previously for the corresponding carboxylated analogues.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Evaluation using latent class models of the diagnostic performances of three ELISA tests commercialized for the serological diagnosis of Coxiella burnetii infection in domestic ruminants.

ELISA methods are the diagnostic tools recommended for the serological diagnosis of Coxiella burnetii infection in ruminants but their respective diagnostic performances are difficult to assess because of the absence of a gold standard. This study focused on three commercial ELISA tests with the following objectives (1) assess their sensitivity and specificity in sheep, goats and cattle, (2) assess the between- and within-herd seroprevalence distribution in these species, accounting for diagnostic errors, and (3) estimate optimal sample sizes considering sensitivity and specificity at herd level. We comparatively tested 1413 cattle, 1474 goat and 1432 sheep serum samples collected in France. We analyzed the cross-classified test results with a hierarchical zero-inflated beta-binomial latent class model considering each herd as a population and conditional dependence as a fixed effect. Potential biases and coverage probabilities of the model were assessed by simulation. Conditional dependence for truly seropositive animals was high in all species for two of the three ELISA methods. Specificity estimates were high, ranging from 94.8% [92.1; 97.8] to 99.2% [98.5; 99.7], whereas sensitivity estimates were generally low, ranging from 39.3 [30.7; 47.0] to 90.5% [83.3; 93.8]. Between- and within-herd seroprevalence estimates varied greatly among geographic areas and herds. Overall, goats showed higher within-herd seroprevalence levels than sheep and cattle. The optimal sample size maximizing both herd sensitivity and herd specificity varied from 3 to at least 20 animals depending on the test and ruminant species. This study provides better interpretation of three widely used commercial ELISA tests and will make it possible to optimize their implementation in future studies. The methodology developed may likewise be applied to other human or animal diseases.

Exposure of Wild Ungulates to the Usutu and Tick-Borne Encephalitis Viruses in France in 2009-2014: Evidence of Undetected Flavivirus Circulation a Decade Ago.

Abstract: Flaviviruses have become increasingly important pathogens in Europe over the past few decades. A better understanding of the spatiotemporal distribution of flaviviruses in France is needed to better define risk areas and to gain knowledge of the dynamics of virus transmission cycles. Serum samples from 1014 wild boar and 758 roe deer from 16 departments (administrative units) in France collected from 2009 to 2014 were screened for flavivirus antibodies using a competitive ELISA (cELISA) technique. Serum samples found to be positive or doubtful by cELISA were then tested for antibodies directed against West Nile virus (WNV), Usutu virus (USUV), Bagaza virus (BAGV), and tick-borne encephalitis/Louping ill viruses (TBEV/LIV) by microsphere immunoassays (except BAGV) and micro-neutralization tests. USUV antibodies were detected only in southeastern and southwestern areas. TBEV/LIV antibodies were detected in serum samples from eastern, southwestern and northern departments. The results indicate continuous circulation of USUV in southern France from 2009 to 2014, which was unnoticed by the French monitoring system for bird mortality. The findings also confirm wider distribution of TBEV in the eastern part of the country than of human clinical cases. However, further studies are needed to determine the tick-borne flavivirus responsible for the seroconversion in southwestern and northern France.

Attachment Patterns of Human and Avian Influenza Viruses to Trachea and Colon of 26 Bird Species - Support for the Community Concept.

Avian influenza A viruses (AIVs) have a broad host range, but are most intimately associated with waterfowl (Anseriformes) and, in the case of the H13 and H16 subtypes, gulls (Charadriiformes). Host associations are multifactorial, but a key factor is the ability of the virus to bind host cell receptors and thereby initiate infection. The current study aims at investigating the tissue attachment pattern of a panel of AIVs, comprising H3N2, H6N1, H12N5, and H16N3, to avian trachea and colon tissue samples obtained from host species of different orders. Virus attachment was not restricted to the bird species or order from which the virus was isolated. Instead, extensive virus attachment was observed to several distantly related avian species. In general, more virus attachment and receptor expression were observed in trachea than in colon samples. Additionally, a human seasonal H3N2 virus was studied. Unlike the studied AIVs, this virus mainly attached to tracheae from Charadriiformes and a very limited set of avian cola. In conclusion, the reported results highlight the importance of AIV attachment to trachea in many avian species. Finally, the importance of chickens and mallards in AIVs dynamics was illustrated by the abundant AIV attachment observed.

Swab cloths as a tool for revealing environmental contamination by Q fever in ruminant farms.

Q fever is a zoonotic abortive disease of ruminants mostly transmitted by inhalation of aerosols contaminated by Coxiella burnetii. Clusters of cases or even epidemics regularly occur in humans but, to date, there is no consensus about the best way to carry out outbreak investigations in order to identify potential farms at risk. Although environmental samples might be useful during such investigations, there are few baseline data on the presence of C. burnetii in the environment of ruminant farms. We thus investigated dust samples from cattle, sheep and goat farm buildings in order to (a) estimate C. burnetii detection frequency and bacterial loads in the environment, and (b) determine whether this environmental contamination is associated with series of abortions attributed to Q fever. We considered 113 herds with a recent abortive episode potentially related (n = 60) or not (n = 53) to C. burnetii. Dust was sampled using a swab cloth and tested by a quantitative PCR method targeting the IS1111 gene. Coxiella burnetii DNA was detected on 9 of 50 cattle farms, 13 of 19 goat farms and 30 of 40 sheep farms. On 16 cloths, bacterial loads were higher than 108 genome equivalents, levels as high as in infectious materials such as placentas and aborted foetuses. Overall, the probability of detecting C. burnetii DNA was higher on small ruminant farms than cattle farms, in herds suspected of Q fever and in large herds. We conclude that swab cloths are a putative indicator of contamination of ruminant farms by C. burnetii.

Characterization of avian influenza virus attachment patterns to human and pig tissues.

Wild birds of Anseriformes and Charadriiformes are natural reservoirs of influenza A viruses (IAVs). Occasionally, IAVs transmit and adapt to mammalian hosts, and are maintained as epidemic strains in their new hosts. Viral adaptions to mammalian hosts include altered receptor preference of host epithelial sialylated oligosaccharides from terminal α2,3-linked sialic acid (SA) towards α2,6-linked SA. However, α2,3-linked SA has been found in human respiratory tract epithelium, and human infections by avian IAVs (AIVs) have been reported. To further explore the attachment properties of AIVs, four AIVs of different subtypes were investigated on human and pig tissues using virus histochemistry. Additionally, glycan array analysis was performed for further characterization of IAVs' receptor structure tropism. Generally, AIV attachment was more abundant to human tissues than to pig tissues. The attachment pattern was very strong to human conjunctiva and upper respiratory tract, but variable to the lower respiratory tract. AIVs mainly attached to α2,3-linked SA, but also to combinations of α2,3- and α2,6-linked SA. The low attachment of these AIV isolates to pig tissues, but high attachment to human tissues, addresses the question whether AIVs in general require passage through pigs to obtain adaptions towards mammalian receptor structures.

Seroprevalence of horses to Coxiella burnetii in an Q fever endemic area.

Coxiella burnetii can infect many animal species, but its circulation dynamics in and through horses is still unclear. This study evaluated horse exposure in an area known to be endemic for ruminants and humans. We assessed antibody prevalence in horse serum by ELISA, and screened by qPCR horse blood, ticks found on horses and dust from stables. Horse seroprevalence was 4% (n = 335, 37 stables) in 2015 and 12% (n = 294, 39 stables) in 2016. Of 199 horses sampled both years, 13 seroconverted, eight remained seropositive, and one seroreverted. Seropositive horses were located close to reported human cases, yet none displayed Q fever-compatible syndromes. Coxiella DNA was detected in almost 40% of collected ticks (n = 59/148 in 2015; n = 103/305 in 2016), occasionally in dust (n = 3/46 in 2015; n = 1/14 in 2016) but never in horse blood. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.

Population genetics, community of parasites, and resistance to rodenticides in an urban brown rat (Rattus norvegicus) population.

Brown rats are one of the most widespread urban species worldwide. Despite the nuisances they induce and their potential role as a zoonotic reservoir, knowledge on urban rat populations remains scarce. The main purpose of this study was to characterize an urban brown rat population from Chanteraines park (Hauts-de-Seine, France), with regards to haematology, population genetics, immunogenic diversity, resistance to anticoagulant rodenticides, and community of parasites. Haematological parameters were measured. Population genetics was investigated using 13 unlinked microsatellite loci. Immunogenic diversity was assessed for Mhc-Drb. Frequency of the Y139F mutation (conferring resistance to rodenticides) and two linked microsatellites were studied, concurrently with the presence of anticoagulant residues in the liver. Combination of microscopy and molecular methods were used to investigate the occurrence of 25 parasites. Statistical approaches were used to explore multiple parasite relationships and model parasite occurrence. Eighty-six rats were caught. The first haematological data for a wild urban R. norvegicus population was reported. Genetic results suggested high genetic diversity and connectivity between Chanteraines rats and surrounding population(s). We found a high prevalence (55.8%) of the mutation Y139F and presence of rodenticide residues in 47.7% of the sampled individuals. The parasite species richness was high (16). Seven potential zoonotic pathogens were identified, together with a surprisingly high diversity of Leptospira species (4). Chanteraines rat population is not closed, allowing gene flow and making eradication programs challenging, particularly because rodenticide resistance is highly prevalent. Parasitological results showed that co-infection is more a rule than an exception. Furthermore, the presence of several potential zoonotic pathogens, of which four Leptospira species, in this urban rat population raised its role in the maintenance and spread of these pathogens. Our findings should stimulate future discussions about the development of a long-term rat-control management program in Chanteraines urban park.

Molecular epidemiology of Coxiella burnetii in French livestock reveals the existence of three main genotype clusters and suggests species-specific associations as well as regional stability.

Q fever is a worldwide zoonosis caused by the bacterium Coxiella burnetii. In domestic ruminants, Q fever main clinical manifestations are abortions. Although the clinical signs may differ between ruminant species, C. burnetii's genetic diversity remains understudied in enzootic areas. Here, we focused on France, where Q fever is enzootic, with the aims to (a) identify potential associations between C. burnetii genotypes and ruminant host species; (b) assess the distribution of C. burnetii genotypes both within French farms and across France's major livestock-farming regions; and (c) suggest a subset of markers for future genotypic studies. We used DNA samples collected between 2006 and 2015 from 301 females (160 cows, 76 ewes, 65 goats) aborted of Q fever within 7 different farming regions. C. burnetii diversity was determined using a multiple-locus variable-number of tandem repeat analysis (MLVA) considering 17 markers. Using a phylogenetic approach, we identified 3 main genotypic clusters divided into 12 sub-clusters. These clusters were significantly associated with ruminant species: almost all the cattle genotypes were found in a "cattle-specific" cluster whereas small ruminants genotypes essentially grouped into the two other clusters. The clusters also proved stable over space and time, some genotypes being more specifically observed in certain farming regions. We also observed some within-farm diversity but this diversity was restricted to a same genotypic cluster. Finally, we identified 6 MLVA markers that maximized the representativeness of the diversity described. Overall, we highlighted that molecular epidemiology is a relevant approach to assess C. burnetii's genetic diversity and to reveal the existence of species-specific associations and regional stability. These results will be valuable in the field to trace genotype circulation among ruminants and from ruminants to humans. Ultimately, the potential links between genotypes and virulence traits need to be investigated to adapt control measures in livestock farms.

High genetic structuring of Tula hantavirus.

Tula virus (TULV) is a vole-associated hantavirus with low or no pathogenicity to humans. In the present study, 686 common voles (Microtus arvalis), 249 field voles (Microtus agrestis) and 30 water voles (Arvicola spec.) were collected at 79 sites in Germany, Luxembourg and France and screened by RT-PCR and TULV-IgG ELISA. TULV-specific RNA and/or antibodies were detected at 43 of the sites, demonstrating a geographically widespread distribution of the virus in the studied area. The TULV prevalence in common voles (16.7 %) was higher than that in field voles (9.2 %) and water voles (10.0 %). Time series data at ten trapping sites showed evidence of a lasting presence of TULV RNA within common vole populations for up to 34 months, although usually at low prevalence. Phylogenetic analysis demonstrated a strong genetic structuring of TULV sequences according to geography and independent of the rodent species, confirming the common vole as the preferential host, with spillover infections to co-occurring field and water voles. TULV phylogenetic clades showed a general association with evolutionary lineages in the common vole as assessed by mitochondrial DNA sequences on a large geographical scale, but with local-scale discrepancies in the contact areas.

The Importance of Ticks in Q Fever Transmission: What Has (and Has Not) Been Demonstrated?

Q fever is a widespread zoonotic disease caused by Coxiella burnetii, a ubiquitous intracellular bacterium infecting humans and a variety of animals. Transmission is primarily but not exclusively airborne, and ticks are usually thought to act as vectors. We argue that, although ticks may readily transmit C. burnetii in experimental systems, they only occasionally transmit the pathogen in the field. Furthermore, we underscore that many Coxiella-like bacteria are widespread in ticks and may have been misidentified as C. burnetii. Our recommendation is to improve the methods currently used to detect and characterize C. burnetii, and we propose that further knowledge of Coxiella-like bacteria will yield new insights into Q fever evolutionary ecology and C. burnetii virulence factors.

The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii.

Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

Laridae: A neglected reservoir that could play a major role in avian influenza virus epidemiological dynamics.

Avian influenza viruses (AIVs) are of great concern worldwide due to their economic impact and the threat they represent to human health. As wild birds are the natural reservoirs of AIVs, understanding AIV dynamics in different avian taxa is essential for deciphering the epidemiological links between wildlife, poultry and humans. To date, only the Anatidae (ducks, geese and swans) have been widely studied. Here, we aim to shed light on the current state of knowledge on AIVs in Laridae (gulls, terns and kittiwakes) versus that in Anatidae by setting forth four fundamental questions: how, when, where and to which host species are AIVs transmitted? First, we describe ecological differences between Laridae and Anatidae and discuss how they may explain observed contrasts in preferential transmission routes and the evolution of specific AIV subtypes. Second, we highlight the dissimilarities in the temporal patterns of AIV shedding between Laridae and Anatidae and address the role that immunity likely plays in shaping these patterns. Third, we underscore that Laridae may be key in promoting intercontinental exchanges of AIVs. Finally, we emphasize the crucial epidemiological position that Laridae occupy between wildlife, domestic birds and humans.

Diversity and global distribution of the Coxiella intracellular bacterium in seabird ticks.

The obligate intracellular bacterium Coxiella burnetii is the etiological agent of Q fever, a widespread zoonotic disease whose most common animal reservoirs are domestic ruminants. Recently, a variety of Coxiella-like organisms have also been reported from non-mammalian hosts, including pathogenic forms in birds and forms without known effects in ticks, raising questions about the potential importance of non-mammalian hosts as reservoirs of Coxiella in the wild. In the present study, we examined the potential role of globally-distributed seabird ticks as reservoirs of these bacteria. To this aim, we tested for Coxiella infection 11 geographically distinct populations of two tick species frequently found in seabird breeding colonies, the hard tick Ixodes uriae (Ixodidae) and soft ticks of the Ornithodoros (Carios) capensis group (Argasidae). We found Coxiella-like organisms in all O. capensis sensu lato specimens, but only in a few I. uriae specimens of one population. The sequencing of 16S rDNA and GroEL gene sequences further revealed an unexpected Coxiella diversity, with seven genetically distinct Coxiella-like organisms present in seabird tick populations. Phylogenetic analyses show that these Coxiella-like organisms originate from three divergent subclades within the Coxiella genus and that none of the Coxiella strains found in seabird ticks are genetically identical to the forms known to be associated with pathogenicity in vertebrates, including C. burnetii. Using this data set, we discuss the potential epidemiological significance of the presence of Coxiella in seabird ticks. Notably, we suggest that these organisms may not be pathogenic forms, but rather behave as endosymbionts engaged in intricate interactions with their tick hosts.

Circulation of a Meaban-like virus in yellow-legged gulls and seabird ticks in the western Mediterranean basin.

In recent years, a number of zoonotic flaviviruses have emerged worldwide, and wild birds serve as their major reservoirs. Epidemiological surveys of bird populations at various geographical scales can clarify key aspects of the eco-epidemiology of these viruses. In this study, we aimed at exploring the presence of flaviviruses in the western Mediterranean by sampling breeding populations of the yellow-legged gull (Larus michahellis), a widely distributed, anthropophilic, and abundant seabird species. For 3 years, we sampled eggs from 19 breeding colonies in Spain, France, Algeria, and Tunisia. First, ELISAs were used to determine if the eggs contained antibodies against flaviviruses. Second, neutralization assays were used to identify the specific flaviviruses present. Finally, for colonies in which ELISA-positive eggs had been found, chick serum samples and potential vectors, culicid mosquitoes and soft ticks (Ornithodoros maritimus), were collected and analyzed using serology and PCR, respectively. The prevalence of flavivirus-specific antibodies in eggs was highly spatially heterogeneous. In northeastern Spain, on the Medes Islands and in the nearby village of L'Escala, 56% of eggs had antibodies against the flavivirus envelope protein, but were negative for neutralizing antibodies against three common flaviviruses: West Nile, Usutu, and tick-borne encephalitis viruses. Furthermore, little evidence of past flavivirus exposure was obtained for the other colonies. A subset of the Ornithodoros ticks from Medes screened for flaviviral RNA tested positive for a virus whose NS5 gene was 95% similar to that of Meaban virus, a flavivirus previously isolated from ticks of Larus argentatus in western France. All ELISA-positive samples subsequently tested positive for Meaban virus neutralizing antibodies. This study shows that gulls in the western Mediterranean Basin are exposed to a tick-borne Meaban-like virus, which underscores the need of exploring the spatial and temporal distribution of this flavivirus as well as its potential pathogenicity for animals and humans.

Flaviviruses in Europe: complex circulation patterns and their consequences for the diagnosis and control of West Nile disease.

In Europe, many flaviviruses are endemic (West Nile, Usutu, tick-borne encephalitis viruses) or occasionally imported (dengue, yellow fever viruses). Due to the temporal and geographical co-circulation of flaviviruses in Europe, flavivirus differentiation by diagnostic tests is crucial in the adaptation of surveillance and control efforts. Serological diagnosis of flavivirus infections is complicated by the antigenic similarities among the Flavivirus genus. Indeed, most flavivirus antibodies are directed against the highly immunogenic envelope protein, which contains both flavivirus cross-reactive and virus-specific epitopes. Serological assay results should thus be interpreted with care and confirmed by comparative neutralization tests using a panel of viruses known to circulate in Europe. However, antibody cross-reactivity could be advantageous in efforts to control emerging flaviviruses because it ensures partial cross-protection. In contrast, it might also facilitate subsequent diseases, through a phenomenon called antibody-dependent enhancement mainly described for dengue virus infections. Here, we review the serological methods commonly used in WNV diagnosis and surveillance in Europe. By examining past and current epidemiological situations in different European countries, we present the challenges involved in interpreting flavivirus serological tests and setting up appropriate surveillance programs; we also address the consequences of flavivirus circulation and vaccination for host immunity.

Recent circulation of West Nile virus and potentially other closely related flaviviruses in Southern France.

In recent years, the number of West Nile virus (WNV) cases reported in horses and humans has increased dramatically throughout the Mediterranean basin. Furthermore, the emergence of Usutu virus (USUV) in Austria in 2001, and its subsequent expansion to Hungary, Spain, Italy, Switzerland, the United Kingdom, and Germany, has given added cause for concern regarding the impact of the spread of flaviviruses on human and animal health in western Europe. Despite frequent detection of WNV and USUV cases in neighboring countries, no case of WNV has been detected in France since 2006 and USUV has never been reported. However, recent investigations focused on detecting the circulation of flaviviruses in France are lacking. We investigated the circulation of WNV and USUV viruses in wild birds in southern France on the basis of a serological survey conducted on a sentinel species, the magpie (Pica pica), in the Camargue area from November, 2009, to December, 2010. We detected WNV-neutralizing antibodies at a high titer (160) in a second-year bird showing recent exposure to WNV, although no WNV case has been detected in humans or in horses since 2004 in the Camargue. In addition, we observed low titers (10 or 20) of USUV-specific antibodies in six magpies, two of which were also seropositive for WNV. Such low titers do not give grounds for concluding that these birds had been exposed to USUV; cross-reactions at low titers may occur between antigenically closely related flaviviruses. But these results urge for further investigations into the circulation of flaviviruses in southern France. They also emphasize the necessity of undertaking epidemiological studies on a long-term basis, rather than over short periods following public health crises, to gain insight into viral dynamics within natural reservoirs.

European H16N3 gull influenza virus attaches to the human respiratory tract and eye.

We explored the attachment of an H16N3 influenza virus to human, mallard, and gull tissues using virus histochemistry applied to tissue microarrays and employing human and mallard viruses as references. Of the viruses tested, the H16N3 gull virus most readily attached to the human respiratory tract and eye. These results underscore the need to assess the potential for gull influenza viruses to replicate in human tissues and further investigate the role of gulls in influenza virus ecology.