RESUMO
Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c+ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression.
Assuntos
Infecções por Bunyaviridae/metabolismo , Leucócitos Mononucleares/virologia , Orthobunyavirus , RNA Viral/metabolismo , Citometria de Fluxo , Imunofluorescência , Genoma Viral/genética , Humanos , Microscopia Confocal , Orthobunyavirus/genética , Orthobunyavirus/metabolismo , Orthobunyavirus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Replicação ViralRESUMO
Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.