Enhanced virulence and neuroinvasion of contemporary Oropouche virus strains in the AG129 mouse model.

Oropouche virus (OROV) is an orthobunyavirus undergoing dramatic re-emergence and rapid geographic expansion across Central and South America, coinciding with the emergence of reassortant lineages carrying novel segment combinations and adaptive mutations. Despite extensive genomic and epidemiological characterization, the biological mechanisms driving OROV resurgence during 2023-2024 remain unclear. Here, we show that contemporary reassortant OROV isolates exhibit markedly enhanced virulence in the AG129 mouse model compared with the historical prototype strain OROVBeAn19991. Two reassortant 2024 isolates-OROVSC2024 from a non-Amazonian region and OROVAC2024 from the Amazon Basin-produced significantly higher viremia, accelerated disease progression, and increased mortality. These isolates disseminated widely, generating substantially elevated viral loads across multiple organs, including the spleen, kidney, liver, lung, uterus, and testes, and-most notably-demonstrated a heightened capacity for the brain. Infection with contemporary reassortant lineages also induced pro-inflammatory responses, characterized by consistent upregulation of IL-1β across tissues. Together, these findings provide experimental evidence that ongoing viral evolution is likely driving a measurable increase in OROV pathogenicity, providing a biological framework to better understand the increased severity of clinical cases reported in recent outbreaks. This work underscores the urgent need for enhanced surveillance, real-time genomic monitoring, and development of targeted countermeasures against this rapidly spreading arbovirus.