Discovery of novel astrovirus and calicivirus identified in ruddy turnstones in Brazil

Birds are the natural reservoir of viruses with zoonotic potential, as well as contributing to the evolution, emergence, and dissemination of novel viruses. In this study, we applied a high-throughput screening approach to identify the diversity of viruses in 118 samples of birds captured between October 2006 to October 2010 in the North and Northeast regions of Brazil. We found nearly complete genomes of novel species of astrovirus and calicivirus in cloacal swabs of ruddy turnstones (Arenaria interpres) collected in Coroa do Avião islet, Pernambuco State. These viruses are positive-sense single-stranded RNA with a genome of ~7 to 8 kb, and were designated as Ruddy turnstone astrovirus (RtAstV) and Ruddy turnstone calicivirus (RTCV), respectively. Phylogenetic analysis showed that RtAstV and RTCV grouped in a monophyletic clade with viruses identified from poultry samples (i.e., chicken, goose, and turkey), including viruses associated with acute nephritis in chickens. Attempts of viral propagation in monkey and chicken cell lines for both viruses were unsuccessful. Also, we found genomes related with viral families that infect invertebrates and plants, suggesting that they might be ingested in the birds' diet. In sum, these findings shed new light on the diversity of viruses in migratory birds with the notable characterization of a novel astrovirus and calicivirus

Novel parvoviruses from wild and domestic animals in Brazil provide new insights into parvovirus distribution and diversity

Parvoviruses (family Parvoviridae) are small, single-stranded DNA viruses. Many parvoviral pathogens of medical, veterinary and ecological importance have been identified. In this study, we used high-throughput sequencing (HTS) to investigate the diversity of parvoviruses infecting wild and domestic animals in Brazil. We identified 21 parvovirus sequences (including twelve nearly complete genomes and nine partial genomes) in samples derived from rodents, bats, opossums, birds and cattle in Pernambuco, São Paulo, Paraná and Rio Grande do Sul states. These sequences were investigated using phylogenetic and distance-based approaches and were thereby classified into eight parvovirus species (six of which have not been described previously), representing six distinct genera in the subfamily Parvovirinae. Our findings extend the known biogeographic range of previously characterized parvovirus species and the known host range of three parvovirus genera (Dependovirus, Aveparvovirus and Tetraparvovirus). Moreover, our investigation provides a window into the ecological dynamics of parvovirus infections in vertebrates, revealing that many parvovirus genera contain well-defined sub-lineages that circulate widely throughout the world within particular taxonomic groups of hosts

Current and future interactions between nature and society

One hundred per cent of the natural units of analysis will continue to be negatively affected, with a concomitant decrease in natures contributions to people, given current trends (business as usual), though the magnitude and exact mechanism of the individual drivers will vary by driver and unit of analysis (established but incomplete){5.4}. For example, tropical moist and dry forest and coastal mangroves will continue to exhibit a decline due to land use change regardless of the scenarios considered, but different local factors (agriculturalization and urbanization, respectively) will be involved (well established) {5.4.1, 5.4.11}. Additionally, some drivers will affect units of analysis differently. Empirical evidence indicates differential effects of climate change: boreal forest is extending northward {5.4.2}, while tundra is diminishing in land area (established but incomplete) {5.4.3}. Thus, some drivers, and their relative roles, will need to be further refined on a local scale and with respect to their proximate factors.2. Multiple drivers will act in synergy and further produce biodiversity loss and impact nature?s contributions to people in most of the units of analysis for the Americas (established but incomplete){5.4}. Climate change, combined with other drivers, is predicted to account for an increasingly larger proportion of biodiversity loss in the future, in both terrestrial and aquatic ecosystems {5.3}. Forest fragmentation, climate change and industrial development increase risk of biodiversity and nature?s contributions to people loss i.e. dry forest unit of analysis {5.4.1.2}. Predictions on invasive species and climate change indicates an increase in habitable areas and their potential impacts on different units of analysis {5.3}.3. Changes in temperature, precipitation regime and extreme climate events are predicted to impact all units of analysis in the Americas (well established) {5.4}. Climate change and the potential impacts on tropical dry forests by changing the frequency of wildfires; change in forest structure and functional composition in the Amazon tropical moist forest; extreme drought events changing nature?s contributions to people in the Amazon region; insect outbreaks and changes in albedo are predicted to significantly impact temperate, boreal and tundra units of analysis, affecting society and indigenous communities and well-being {5.4}.4. Thresholds, or tipping points (conditions resulting in rapid and potentially irreversible changes) may have already been exceeded for some ecosystems and are likely for others (established but incomplete). For instance, it is considered more likely than not that such a threshold has already been passed in the cryosphere with respect to summer sea ice (established but incomplete) {5.4.12}. Model simulations indicate changes in forest structure and species distribution in the Amazon forest in response to global warming and change in precipitation patterns (forest die-back) (established but incomplete) {5.4.1}. So too, a 4oC increase in global temperatures is predicted to likely cause widespread die off of boreal forest due to greater susceptibility to disease {5.4.2} and global temperature increases may have already started persistent thawing of the permafrost {5.4.3}. Under 4°C warming, widespread coral reef mortality is expected with significant impacts on coral reef ecosystems {5.4.11}. Sea surface water temperature increase will cause a reduction of sea grass climatic niche: those populations under seawater surface temperature thresholds higher than the temperature ranges required by the species could become extinct by 2100 with concomitant loss of ecosystem services.IPBES/6/INF/4/Rev.15415. Changes in nature and nature?s contributions to people in most units of analysis are increasingly driven by causal interactions between distant places (i.e. telecouplings) (well established) {5.6.3}, thus scenarios and models that incorporate telecouplings will better inform future policy decisions. Nature and nature?s contributions to people in telecoupled systems can be affected negatively or positively by distant causal interactions. Provision of food and medicine from wild organisms in temperate and tropical grasslands, savannas and forests of South America is being dramatically reduced due to land-use changes driven by the demand of agricultural commodities (e.g. soybeans) mainly from Europe and China. Conservation of insectivorous migratory bats in Mexico benefits pest control in agroecosystems of North America, resulting in increased yields and reduced pesticide costs. Trade policies and international agreements will thus have an increasingly strong effect on environmental outcomes in telecoupled systems.6. Policy interventions have resulted in significant land use changes at the local and regional scales and will continue to do so through 2050. These policies have affected nature?s contributions to people both positively and negatively, and provide an opportunity to manage trade-offs among nature?s contributions to people (well established) {5.4}. Land use changes are now mainly driven by high crop demand, big hydropower plans, rapid urban growth and result in a continued loss of grasslands {5.4.4, 5.4.5}. However, strategies for establishing conservation units have helped in reducing deforestation in the Brazilian Amazon from the period of 2004 to 2011 (well established) {5.4.1}. Similarly, wetland protection policies and regulation have helped reduce the conversion of wetlands in North America {5.4.7}. Policies based on command and control measures may be limited in providing effective reduction in ecosystem loss and should be complemented with policies acknowledging multiple values {5.6.3}.7. Policy interventions at vastly differing scales (from national to local) lead to successful outcomes in mitigating impacts to biodiversity (established but incomplete){5.4}. For instance, long-established governmental protections of wetlands in North America have significantly slowed and may have stopped wetland loss based on acreage {5.4.7}. In South America, where mangrove loss continues at a rate of one to two per cent, different stakeholders such as local communities and/or governments have been successful in protecting mangroves based on empowerment and shared interests in their preservation {5.4.11}.8. Pressures to nature are projected to increase by 2050, negatively affecting biodiversity as indicated by a potential reduction of the mean species abundance index. However, the magnitude of the pressures by 2050 are expected to be less under transition pathways to sustainability in comparison to the business as usual scenario (established but incomplete), {5.5}. The Global Biodiversity model projected that under the business as usual scenario mean species abundance had decreased in the Americas by approximately 30 per cent by 2010 compared to its values prior to European settlement of the New World, with historical losses primarily attributed to land transformation to agricultural uses. Using the Global Biodiversity model, there is an additional projected loss of 9.6 per cent by 2050, primarily attributed to some additional land use changes , and especially to climate change, which will steadily increase relative to other drivers considered in the model. However, under the transition pathways to sustainability of global technologies, decentralised solutions, and consumption change pathways, the projected losses are 6 per cent, 5 per cent, and 5 per cent, respectively,IPBES/6/INF/4/Rev.1542achieving a relative improvement of approximately 30 per cent to 50 per cent compared to the business as usual scenario. Under these pathways, climate change mitigation, the expansion of protected areas and the recovery of abandoned lands would significantly contribute to reducing biodiversity loss.9. Participative scenarios have proven to be a successful tool for envisioning potential futures and pathways and to embrace and integrate multiple and sometime conflicting values and their role in promoting bottom-up decision making in the face of futures uncertainties (well established) {5.3}. The use of participative approaches to develop scenarios has increased during recent years in the Americas. The inclusion of different stakeholders and their knowledges in the process of constructing potential futures has promoted a better understanding of the complexity of the social-ecological systems in which they are embedded. This has enhanced co-learning processes between all actors involved, even those normally under-represented in decision-making activities. As a result, several participative scenario exercises have motivated community-based solutions and local governance initiatives all pointing towards the development of adaptive management strategies {5.3}.10. Pathways that consider changes in societal options will lead to less pressure to nature (established but incomplete) {5.6.3}. An example is the indirect impact that shifts in urban dietary preferences have on agricultural production and expansion, and food options that are expected to continue growing into the future. Therefore, not only is there a strong connection between urbanization and economic growth, but also between affluence (and urban preferences) and the global displacement of land use particularly from high-income to low-income countries.11. Available local studies informing regional futures of nature and natures benefit to people do not allow scalability as of yet (well established) {5.3}. The challenge in expanding the findings from local studies resides in the fact that a number of comparable local studies are still not available. Information is scattered throughout the region by the use of different units, methods and scales, which prevents a local-to-regional generalization. The list of nature indicators used in studies at local scales is large and heterogeneous (well established). Even for the same indicator (e.g. biodiversity), different metrics are used (e.g. species-area curve, mean species abundance) {5.5}. In other cases, multiple indicators are used to describe different aspects of biodiversity and ecosystem services. In this latter case, synergies and trade-offs are explicitly mentioned with a clear pattern in which increasing the provision of some indicators result in the detriment of others {5.3}. For example, agriculture expansion leading to loss in biodiversity illustrates a common trend from local studies expected to continue into the future.12. There is a significant research gap in the development of models and scenarios that integrate drivers, nature, natures contributions to people and good quality of life (well established){5.3}. Models and scenarios can be powerful tools to integrate and synthesize the complex dynamics of coupled human and nature systems, and to project their plausible behaviors into the future. Most existing models and scenarios focus on the link between drivers and its impacts on nature. Few cases exist in which models or scenarios integrate the relationships between changes in nature and changes in natures contributions to people and good quality of life {5.3}. Inter-and trans-disciplinary modeling efforts will be required to address this research gap {5.3}.Fil: Klatt, Brian. Michigan State University; Estados UnidosFil: Ometto, Jean Pierre. National Institute For Space Research; BrasilFil: García Marquez, Jaime. Universität zu Berlin; AlemaniaFil: Baptiste, María Piedad. Instituto Alexander Von Humboldt; ColombiaFil: Instituto Alexander von Humboldt. Independent Consultant; CanadáFil: Acebey, Sandra Verónica. No especifíca;Fil: Guezala, María Claudia. Inter-american Institute For Global Change Research; PerúFil: Mastrangelo, Matias Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pengue, Walter Alberto. Universidad Nacional de General Sarmiento; ArgentinaFil: Blanco, Mariela Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Saavedra 15. Centro de Estudios e Investigaciones Laborales; ArgentinaFil: Gadda, Tatiana. Universidade Tecnológica Federal Do Paraná; BrasilFil: Ramírez, Wilson. Instituto Alexander Von Humboldt; ColombiaFil: Agard, John. University Of West Indies; Trinidad y TobagoFil: Valle, Mireia. Universidad Laica Eloy Alfaro de Manabí; Ecuado

Detection of hantavirus in bats from remaining rain forest in São Paulo, Brazil

Background\ud The significant biodiversity found in Brazil is a potential for the emergence of new zoonoses. Study in some places of the world suggest of the presence to hantavirus in tissues of bats. Researches of hantavirus in wildlife, out rodents, are very scarce in Brazil. Therefore we decided to investigate in tissues of different species of wild animals captured in the same region where rodents were detected positive for this virus. The present work analyzed ninety-one animals (64 rodents, 19 opossums, and 8 bats) from a region of the Atlantic forest in Biritiba Mirin City, São Paulo State, Brazil. Lungs and kidneys were used for RNA extraction.\ud \ud Findings\ud The samples were screened for evidence of hantavirus infection by SYBR-Green-based real-time RT-PCR. Sixteen samples positive were encountered among the wild rodents, bats, and opossums. The detection of hantavirus in the lungs and kidneys of three marsupial species (Micoureus paraguayanus, Monodelphis ihering, and Didelphis aurita) as well in two species of bats (Diphylla ecaudata and Anoura caudifer) is of significance because these new hosts could represent an important virus reservoirs.This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESP. The field support and animals identification was made at Museum of Zoology, Universidade de Sao Paulo, USP and Universidade Bandeirante, UNIBAN, Brazil

Serological and molecular investigation of viral agents in free-living jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência – RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onças-pintadas nesta região.This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest – SFIMT and Rapid Fluorescent Focus Inhibition Test – RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region

Avian influenza virus (H11N9) in migratory shorebirds wintering in the Amazon region, Brazil

Aquatic birds are the natural reservoir for avian influenza viruses (AIV). Habitats in Brazil provide stopover and wintering sites for water birds that migrate between North and South America. The current study was conducted to elucidate the possibility of the transport of influenza A viruses by birds that migrate annually between the Northern and Southern Hemispheres. In total, 556 orotracheal/cloacal swab samples were collected for influenza A virus screening using real-time RT-PCR (rRT-PCR). The influenza A virus-positive samples were subjected to viral isolation. Four samples were positive for the influenza A matrix gene by rRT-PCR. From these samples, three viruses were isolated, sequenced and characterized. All positive samples originated from a single bird species, the ruddy turnstone (Arenaria interpres), that was caught in the Amazon region at Caeté Bay, Northeast Pará, at Ilha de Canelas. To our knowledge, this is the first isolation of H11N9 in the ruddy turnstone in South America. (Résumé d'auteur

Diagnóstico brasileiro sobre biodiversidade e serviços ecossistêmicos : sumário para tomadores de decisão

A biodiversidade e os ecossistemas são elementos importantes para enfrentar as crises socioeconômicas e ambientais nacionais e globais, uma vez que proporcionam novas oportunidades de desenvolvimento. Incorporar a biodiversidade na vida cotidiana é uma oportunidade de ouro para promover o uso sustentável da biodiversidade e dos serviços ecossistêmicos. A conservação e a restauração da biodiversidade, dos ecossistemas e de seus serviços associados mostram potencial para um novo desenvolvimento social e econômico, como fonte de geração de emprego e renda, redução da pobreza e da desigualdade socioeconômica. A diversidade biológica brasileira também se expressa em sua imensa diversidade cultural, com uma grande variedade de detentores de conhecimento indígenas e tradicionais. Esses povos possuem vasto conhecimento sobre agrobiodiversidade, pesca, manejo do fogo, medicina natural, entre outros de valor comercial, cultural e espiritual. As principais conclusões deste Sumário para Tomadores de Decisão é que as mudanças no uso da terra e as mudanças climáticas tenham sido - e continuarão sendo ao longo deste século - os principais vetores da perda de biodiversidade e serviços ecossistêmicos no país. Intervenções políticas em diferentes níveis (do local ao nacional, do público ao privado) e a aplicação das leis existentes (mecanismos regulatórios e incentivos) são necessárias para promover a mitigação dos impactos negativos sobre a biodiversidade e a perda de serviços ecossistêmicos. O Brasil já possui uma ampla variedade de instrumentos de política e opções de governança socioambiental, bem como compromissos globais (ODS, Metas de Aichi, Acordo de Paris) relacionados à possibilidade de um futuro sustentável. Entretanto, o controle ineficiente da gestão ou a falta de incentivo para cumprir as regras traz riscos para a consolidação do caminho para esse futuro. O país tem instituições fortes e capazes, mas problemas de infraestrutura, processos lentos, medidas ineficazes e conflitos judiciais, sociais e ecológicos impedem a realização de um desempenho eficiente. Há uma falta de comunicação entre a ciência e a sociedade que precisa ser melhorada por meio do estabelecimento de um fluxo efetivo que torne a comunicação inclusiva e representativa, alcançando os tomadores de decisão públicos e privados. Esforços permanentes para integrar essas duas esferas de conhecimento na sociedade são desejáveis para criar confiança entre os formuladores de políticas e os pesquisadores.Biodiversity and ecosystems are important elements for addressing national and global socioeconomic and environmental crises, since they provide new development opportunities, for example, as source of job and income creation, and reduction in poverty and socioeconomic inequity. Brazilian biological diversity is also expressed in its immense cultural diversity, with a great variety of knowledge holders. These peoples possess vast knowledge on agrobiodiversity, fishing, fire management, natural medicine, among others of commercial, cultural and spiritual value. The main conclusions of this Summary for Police Makers is that land use changes and climate changes have been - and will continue to be throughout this century - the main drivers that result in the loss of biodiversity and ecosystem services in the country. Political interventions at different levels (from local to national, from public to private) and the enforcement of existing laws (regulatory mechanisms and incentives) are required to cope with the mitigation of the negative impacts of biodiversity and ecosystem services loss. Brazil has already a wide variety of policy instruments and socioenvironmental governance options, as well as global commitments (ODS, Aich Targets, Paris Agreement) related to the objective of a sustainable future. However, inefficient management control or lack of incentive to comply with the rules pose risks to consolidating the path to this future. The country has strong and capable institutions, but infrastructural problems, slow processes, inefficient measurements and judicial, social and ecological conflicts obstruct a proficient performance. There is a lack of communication between science and society which needs to be improved by establishing an effective flow that makes communication inclusive and representative, reaching public and private decision makers. Permanent efforts to integrate Science and policy knowledges are desirable to build confidence between policy makers and researchers

Temporal abundance of Aedes aegypti in Manaus, Brazil, measured by two trap types for adult mosquitoes

A longitudinal study was conducted in Manaus, Brazil, to monitor changes of adult Aedes aegypti (L.) abundance. The objectives were to compare mosquito collections of two trap types, to characterise temporal changes of the mosquito population, to investigate the influence of meteorological variables on mosquito collections and to analyse the association between mosquito collections and dengue incidence. Mosquito monitoring was performed fortnightly using MosquiTRAPs (MQT) and BG-Sentinel (BGS) traps between December 2008-June 2010. The two traps revealed opposing temporal infestation patterns, with highest mosquito collections of MQTs during the dry season and highest collections of BGS during the rainy seasons. Several meteorological variables were significant predictors of mosquito collections in the BGS. The best predictor was the relative humidity, lagged two weeks (in a positive relationship). For MQT, only the number of rainy days in the previous week was significant (in a negative relationship). The correlation between monthly dengue incidence and mosquito abundance in BGS and MQT was moderately positive and negative, respectively. Catches of BGS traps reflected better the dynamic of dengue incidence. The findings help to understand the effects of meteorological variables on mosquito infestation indices of two different traps for adult dengue vectors in Manaus

Surveillance of West Nile virus in Brazil.

O Vírus do Nilo Ocidental, do inglês West Nile virus (WNV) é um patógeno emergente que é amplamente distribuído na América do Norte e Central. A recente introdução na América do Sul chamou a atenção para a propagação do WNV em países Latino Americanos. O ciclo de transmissão envolve mosquitos, pássaros, cavalos e seres humanos. A avaliação sorológica realizada nestes estudo foi composta por 678 soros de equídeos e 478 soros de aves, realizada por meio do ensaio ELISA de bloqueio específico para WNV e somente as amostras com resultados positivos foram confirmadas por testes de neutralização por redução em placas (PRNTs). A análise molecular foi realizada em soros de 1.241 equídeos saudáveis e em 63 macerados de cérebros de equídeos que morreram de encefalite e obtiveram resultados previamente negativos para outros patógenos. Também testamos swabs de 3.445 aves pelo método molecular, além de amostras de 24 morcegos e 11 onças. As amostras analisadas foram coletadas em diferentes biomas do Brasil. Identificamos pelo ELISA anticorpos para o WNV em treze equídeos e cinco pássaros e o teste de PRNT90 confirmou positividade para o WNV em quatro amostras de equídeos coletadas em 2009 em uma região entre a Amazônia e o Pantanal. Nenhuma das amostras de aves positivas pelo ELISA foram confirmadas por PRNT90. Das 4.784 amostras testadas por RT-PCR, penas duas apresentaram resultados positivos para a detecção, sendo uma ave residente na região do Pantanal e um anatídeo na região do Maranhão, respectivamente. A circulação do WNV é confirmada pela presente pesquisa em larga escala, mesmo na ausência da detecção de casos clínicos.West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 1241 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 3.445 birds, 24 bats and 11 phanteras. The samples analysed were collected in different biomes of Brazil. We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90. Of the 4.784 samples tested by RT-PCR, only two were positive for the detection, a resident bird in the Pantanal region and a duck in the region of Maranhão, respectively. WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases