Selective pressure-driven identification of conserved regions for Oropouche virus molecular detection

Oropouche virus (OROV) is an emerging virus that causes a wide spectrum of febrile illnesses in humans. In this study, we examined two of the most commonly used primer sets for the clinical diagnosis of OROV to identify potential reduction in diagnostic sensitivity or even escape due to genetic variability in the target regions of circulating strains. We also evaluated selective pressures on the S segment to determine the optimal region for selecting diagnostic targets. For this, a total of 225 OROV S segment sequences (2015-2024) from multiple American countries were retrieved from NCBI and aligned with commonly used RT-PCR primers to identify mismatches. Subsequently, sequences (1960-2024) were analyzed for evolutionary studies, using HyPhy (Datamonkey), applying aBSREL, MEME, and FUBAR to assess branch- and site-level selection. Alignment of 225 OROV S segment sequences revealed widespread mismatches in primer binding regions. Selection pressure analysis showed no branch-level positive selection, one site under episodic diversifying selection, and 81 sites under purifying selection. Notably, highly conserved regions did not overlap with commonly targeted primer sites. These findings suggest that current diagnostic assays may be vulnerable to reduced sensitivity due to genetic variability. Continuous genomic surveillance and comprehensive primer design studies are essential to ensure reliable detection of emerging OROV strains.