Protocol for detection of Oropouche viruses from human serum.

Oropouche virus (OROV), an arthropod-borne virus, has reemerged in the Americas. In 2023-2024, the largest quantitative reverse-transcription PCR (RT-qPCR)-confirmed outbreak of OROV was reported and was associated with severe clinical outcomes for the first time. Moreover, a dramatic increase in cases is unfolding in early 2025. Here, we provide a protocol for detecting OROV from human serum. We describe steps for viral isolation, titration, and an in-house RT-qPCR validated in the field. We then detail procedures for measurement of anti-OROV-neutralizing antibodies. For complete details on the use and execution of this protocol, please refer to Ciuoderis et al.(1) and Usuga et al.(2).