Abstract
Rocio virus (ROCV), a neurotropic arbovirus of the Orthoflavivirus genus, caused Brazil’s largest encephalitis outbreak in the 1970s but has since been rarely detected. We report the first ROCV encephalitis clinical case after nearly 40 years and isolated and sequenced the virus from CNS tissue of a horse. Metatranscriptomics enabled full-genome sequencing, revealing divergence from the reference sequence at an evolutionary rate of 5.8–7.0 × 10⁻⁴, consistent with Orthoflavivirus. Thirty-one non-synonymous substitutions were identified, with the envelope protein most affected. Findings indicate ROCV cryptic circulation and ongoing evolution. Horses may act as sentinels, underscoring the need for strengthened ROCV genomic surveillance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00705-026-06574-9.