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CNPq (440685/2016-8, 421598/2018-2), by CAPES (88887.130716/ 2016-00), by the Pan American Health Organization (IOC-007-FEX-19-2-2-30), by the Fundac?o Carlos Chagas Filho de Amparo ? Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant number E-26/2002.930/2016 by the European Union?s Horizon 2020 Research and Innovation Programme under ZIKAlliance Grant Agreement no. 734548, by the Horizon 2020 through ZikaPlan and ZikAction (grant agreement numbers 734584 and 734857) and by the National Institutes of Health USA grant U01 AI151698 for the United World Antiviral Research Network (UWARN). Funda??o de Amparo ? Pesquisa do Estado do Rio de Janeiro (FAPERJ). Department of Zoology, University of Oxford. Universidade Federal de Minas Gerais. Departamento de Medicina Veterin?ria Preventiva. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Flaviv?rus. Rio de Janeiro, RJ, Brazil / Universidade Federal de Minas Gerais. Laborat?rio de Gen?tica Celular e Molecular. Belo Horizonte, MG, Brazil. Universidade Federal do Piau?. Departamento De Morfofisiologia Veterin?ria. Teresina, PI, Brazil. Universidade Federal de Minas Gerais. Laborat?rio de Gen?tica Celular e Molecular. Belo Horizonte, MG, Brazil / University of KwaZulu-Natal. College of Health Sciences. School of Laboratory Medicine and Medical Sciences. KwaZulu-Natal Research Innovation and Sequencing Platform. Durban, South Africa / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Coordena??o Geral dos Laborat?rios de Sa?de P?blica. Bras?lia, DF, Brazil. Universidade Federal de Minas Gerais. Laborat?rio de Gen?tica Celular e Molecular. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Flaviv?rus. Rio de Janeiro, RJ, Brazil. Universidade Federal de Minas Gerais. Laborat?rio de Gen?tica Celular e Molecular. Belo Horizonte, MG, Brazil. Funda??o Ezequiel Dias. Laborat?rio Central de Sa?de P?blica. Belo Horizonte, MG, Brazil. Funda??o Municipal de Sa?de. Diretoria de Vigil?ncia em Sa?de. Teresina, PI, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil. Universidade Federal de Minas Gerais. Departamento de Medicina Veterin?ria Preventiva. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Departamento de Medicina Veterin?ria Preventiva. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Departamento de Medicina Veterin?ria Preventiva. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais - Campus Pampulha. Escola de Veterin?ria. Hospital Veterin?rio. Setor de Cl?nica de Equinos. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Flaviv?rus. Rio de Janeiro, RJ, Brazil. Universidade Federal de Santa Maria. Department of Large Animal Clinic. Rio Grande do Sul, RS, Brazil. University of KwaZulu-Natal. College of Health Sciences. School of Laboratory Medicine and Medical Sciences. KwaZulu-Natal Research Innovation and Sequencing Platform. Durban, South Africa. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Coordena??o Geral das Arboviroses. Bras?lia, DF, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Coordena??o Geral das Arboviroses. Bras?lia, DF, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Coordena??o Geral das Arboviroses. Bras?lia, DF, Brasil. Organiza??o Pan-Americana da Sa?de, Organiza??o Mundial da Sa?de. Bras?lia, DF, Brazil. Universidade Federal do Piau?. Centro de Ci?ncias Agr?rias. Departamento de Cl?nica e Cirurgia Veterin?ria. Teresina, PI, Brazil. Universidade Vale do Para?ba. Faculdade de Ci?ncias da Sa?de. Medicina Veterin?ria. S?o Jos? Dos Campos, SP, Brazil. Universidade Federal de Minas Gerais. Departamento de Cl?nica e Cirurgia Veterin?rias. Escola de Veterin?ria. Belo Horizonte, MG, Brazil. Universidade Federal do Piau? - Campus Prof. Cinobelina Elvas. Laborat?rio de Gen?tica e Conserva??o de Germoplasma. Bom Jesus, Piau?, PI, Brazil. Universidade Federal do Piau? - Campus Prof. Cinobelina Elvas. Laborat?rio de Gen?tica e Conserva??o de Germoplasma. Bom Jesus, Piau?, PI, Brazil. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Flaviv?rus. Rio de Janeiro, RJ, Brazil. Oswaldo Cruz Foundation. Coordena??o dos Laboratorios de Referencia. Rio de Janeiro, RJ, Brazil. University of Oxford. Department of Zoology. Oxford, OX, UK. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Laborat?rio de Flaviv?rus. Rio de Janeiro, RJ, Brazil / Universidade Federal de Minas Gerais. Laborat?rio de Gen?tica Celular e Molecular. Belo Horizonte, MG, Brazil. Background: West Nile virus (WNV) was first sequenced in Brazil in 2019, when it was isolated from a horse in the Esp?rito Santo state. Despite multiple studies reporting serological evidence suggestive of past circulation since 2004, WNV remains a low priority for surveillance and public health, such that much is still unknown about its genomic diversity, evolution, and transmission in the country. Methods: A combination of diagnostic assays, nanopore sequencing, phylogenetic inference, and epidemiological modeling are here used to provide a holistic overview of what is known about WNV in Brazil. Results: We report new genetic evidence of WNV circulation in southern (Minas Gerais, S?o Paulo) and northeastern (Piau?) states isolated from equine red blood cells. A novel, climate-informed theoretical perspective of the potential transmission of WNV across the country highlights the state of Piau? as particularly relevant for WNV epidemiology in Brazil, although it does not reject possible circulation in other states. Conclusion: Our output demonstrates the scarceness of existing data, and that although there is sufficient evidence for the circulation and persistence of the virus, much is still unknown on its local evolution, epidemiology, and activity. We advocate for a shift to active surveillance, to ensure adequate preparedness for future epidemics with spill-over potential to humans.

We report genetic evidence of WNV circulation from southern and northeastern Brazilian states isolated from equine red blood cells. In the northeastern state the tenth human case was also detected, presenting neuroinvasive disease compatible with WNV infection. Our analyses demonstrate that much is still unknown on the virus’ local epidemiology. We advocate for a shift to active surveillance, to ensure adequate control for future epidemics with spill-over potential to humans.

West Nile virus (WNV) is a neurovirulent mosquito-borne Flavivirus that is maintained in nature by a zoonotic transmissioncycle between avian hosts and ornithophilic mosquito vectors, mostly from the Culex genus. Until the 1990s, WNV wasconsidered to be an old-world arbovirus, but in 1999, WNV emerged in the United States (US) and spread rapidly, becoming amajor threat to public health. WNV adapted to the transmission cycle involving American mosquitoes and birds and reachedCentral and South America in subsequent years. In 2003, the National West Nile Fever Surveillance System was created in Brazilbased on serological screening of animals and sentinel vectors, as recommended by the Pan American Health Organization(PAHO) and the World Health Organization (WHO). Since 2008, serological evidence of WNV infection in Brazilian horseshas been reported, and the circulation of WNV has been monitored through the regular serological screening of sentinel horsesand reporting of encephalomyelitis cases. Horses are highly susceptible to WNV infection, and outbreaks of neurologicaldisease among horses often precede human cases. In this regard, equine surveillance has been essential in providing earlywarning to public and animal health authorities in several countries, including Brazil. This demonstrates the need for animaland public health intervention programs to allocate resources to make veterinarians aware of the role they can play in thehuman surveillance processes by monitoring horses. This review discusses the importance of equine surveillance and the gapthat veterinarians can fill on the front line in human surveillance, in Brazil and worldwide, in the context of “One Health”