The Oropouche virus (OROV) is a segmented negative-sense RNA arbovirus member of the Peribunyaviridae family, associated with recurring epidemics of Oropouche fever in Central and South America. Since its identification in 1955, OROV has been responsible for outbreaks in both rural and urban areas, with transmission involving sylvatic and urban cycles. This study focuses on the characterization of an OROV isolate from a human clinical sample collected in the state of Rio de Janeiro, a non-endemic region in Brazil, highlighting ultrastructural and morphological aspects of the viral replicative cycle in Vero cells. OROV was isolated in Vero cell monolayers which, following viral inoculation, exhibited marked cytopathic effects (CPEs), mainly represented by changes in cell morphology, including membrane protrusions and vacuolization, as well as cell death. Studies by transmission electron microscopy (TEM) revealed significant ultrastructural changes, such as apoptosis, intense remodeling of membrane-bound organelles and signs of rough endoplasmic reticulum and mitochondrial stress. Additionally, the formation of specialized cytoplasmic vacuoles and intra- and extracellular vesicles emphasized trafficking and intercellular communication as essential mechanisms in OROV infection. RT-qPCR studies confirmed the production of viral progeny in high titers, corroborating the efficiency of this experimental model. These findings contribute to a better understanding of the cytopathogenic mechanisms of OROV infection and the contribution of cellular alterations in OROV morphogenesis.

Ilhéus Virus (ILHV) was first detected in 1944 in Ilhéus, state of Bahia, northeast Brazil. During cellular infection, orthoflaviviruses induce cellular changes related both to the replication process, the formation of replication complexes, and to structures resulting from cellular damage. Although more detailed data are available in the literature for other orthoflaviviruses, the relationship between ILHV, the formation of these structures, its replication cycle, and cellular changes remains unknown. One of the main objectives of this study is to characterize the primary ultrastructural changes in green monkey kidney epithelial cell lineage (Vero cell) infected with ILHV, as well as to map its replication cycle, virion structure, and genome. To achieve these objectives, Vero cell monolayers were infected with an MOI of 0.01 and collected at different times post-infection. Cell monolayers were evaluated under bright-field microscopy and transmission electron microscopy. Ultrastructural analyses confirmed that ILHV can induce the formation of double-membrane vesicles, convoluted membranes, and vesicular packets. These structures, like those observed in zika (ZIKV) and dengue (DENV) viruses, form replication complexes that aid ILHV’s replication process in cells. Our preliminary results reveal that ILHV infection induces cytopathogenesis like that observed in vitro studies for other arboviruses.