Phenotypic Characterization of Historical and Contemporary Oropouche Virus Strains Reveals Divergent Replication Dynamics in Multiple Cell Lines

Oropouche virus (OROV), a significant public health threat in Latin America, has recently expanded beyond its endemic region by the emergence of a recombinant lineage. This study aimed to characterize the replication dynamics of a historical (BeAn19991) and two contemporary (AM0088, OROV43) OROV isolates in mammalian and mosquito cell lines. Using multi- and one-step growth curves, plaque assays, and strand-specific qPCR, we demonstrated that contemporary isolates exhibit distinct phenotypic profiles. While the prototype BeAn19991 displayed robust, sustained replication yielding high infectious titers, contemporary isolates showed faster kinetics with earlier peak production followed by a decline. A significant finding was a major asynchrony between genomic RNA synthesis and the production of infectious particles for isolates AM0088 and OROV43. Strand-specific qPCR revealed excessively high levels of viral RNA relative to much lower infectious titers, particularly in mosquito cells, suggesting the generation of defective interfering particles. These results indicate that contemporary OROV isolates have evolved a replication strategy characterized by rapid initial growth and potentially enhanced transmission. This phenotypic shift likely contributes to the virus' increasing incidence and geographical spread, underscoring the need for continued surveillance and research into its evolution. • Contemporary OROV isolates show faster, earlier replication peaks than the historical prototype. • Major asynchrony between RNA synthesis and infectious particle production in recent isolates. • High RNA-to-PFU ratios suggest defective interfering particles in contemporary OROV strains. • Phenotypic shift may enhance transmission and explain OROV's recent geographical spread.