Alphavirus madariaga-associated encephalomyelitis in equines from the Brazilian Pantanal reveals arbovirus circulation in a peri-urban environment.

Alphavirus madariaga (MADV), a member of the eastern equine encephalitis virus complex, is an emerging arbovirus in South America, with limited information regarding its occurrence and molecular characterization in equine populations. This study reports the detection and genetic characterization of MADV in equines from the state of Mato Grosso, Brazil. Two horses presenting acute neurological disease with a clinical course of approximately two days were evaluated. Histopathological examination revealed nonsuppurative meningoencephalomyelitis. Molecular screening for neurotropic viruses using TaqMan RT-qPCR assays was negative for rabies virus, EHV-1, WEEV, VEEV, WNV, and flaviviruses, while MADV was detected with Ct values of 27.9 and 31.4. Partial NS1 and E1 gene fragments were amplified and sequenced. Phylogenetic analyses based on both genes consistently placed the strain, named Santo Antonio, within the MADV genotype III clade, with strong support in the E1 analysis. The cases occurred in a peri-urban area located near Santo Antônio do Leverger, approximately 30 km from Cuiabá, highlighting a potential interface between sylvatic and human-modified environments. These findings provide molecular evidence of MADV circulation in central-western Brazil and association with equines disease. Our results reinforce the role of horses as sentinels for neurotropic arbovirus surveillance before reaching human population.