Abstract
Oropouche virus (OROV), an arthropod-borne virus transmitted by Culicoides paraensis, is an endemic arbovirus that historically circulates mostly in the Amazon basin. Between 2022 and 2024, it reemerged as a more widespread public health concern in South America. We conducted a pooled-sample molecular surveillance study to understand the prevalence of Oropouche fever in Brazil's southernmost state. Over 18 months, we analyzed 4060 samples to monitor the virus emergence in the Rio Grande do Sul state. We detected the first human case of OROV in the state, and our phylogenetic reconstruction indicated a travel-related introduction from the Amazon region into Rio Grande do Sul. Despite the absence of local transmission, the invasion of Culicoides paraensis and enzootic circulation of the OROV in Rio Grande do Sul highlight the risk of Oropouche fever outbreaks in the region. We demonstrated that pooled-sample surveillance effectively monitors virus introduction during periods of low endemic circulation, serving as an essential active surveillance tool for the timely detection of virus emergence and enhancing public health preparedness. The multiple introductions of distinct OROV lineages into southern Brazil underscore the importance of genomic surveillance and public health strategies to monitor and mitigate arbovirus spread in the region.