On 27 May 2024, the Cuban Ministry of Health reported the first outbreak of Oropouche fever on the island. The etiologic agent, Oropouche virus (OROV), is a poorly understood arbovirus that has been known since the 1960s and represents a public health burden in Latin America. We report the whole-genome characterization of the first European OROV isolate from a returning traveler from Cuba with Oropouche fever-like symptoms. The isolate was obtained from the patient’s serum; whole-genome sequencing was performed by next-generation sequencing, followed by phylogenetic analysis and genetic variability studies. The analysis showed that the most closely related sequence was from the French Guiana 2020 outbreak. Interestingly, our isolate is a reassortant virus, included in a highly supported monophyletic clade containing recent OROV cases (Brazil 2015–Colombia 2021), separated from the other four previously known genotypes. More deeply, it was found to be included in a distinct branch containing the sequences of the Brazil 2022–2024 outbreak. The reassortment event involved the S and L segments, which have high similarity with sequences belonging to a new cluster (here defined as OROV_SCDC_2024), while the M segment shows high similarity with older sequences. These results likely describe the viral strain responsible for the current outbreak in Cuba, which may also reflect the ongoing outbreak in Latin America. Further studies are needed to understand how OROV evolves towards traits that facilitate its spread and adaptation outside its original basin, and to track its spread and evolution in the European continent.

Recurrent mpox outbreaks caused by the monkeypox virus (MPXV) have prompted the World Health Organization to declare a Public Health Emergency of International Concern and have stimulated the development of medical interventions. Here, we used antigen-agnostic identification of MPXV-neutralizing monoclonal antibodies from convalescent or vaccinated people, AlphaFold 3–based predictive modeling, and cryo–electron microscopy to identify the protein encoded by orthopoxviral gene (OPG) 153 (MPXV A28) as a target of broadly neutralizing antibodies. OPG153-targeting antibodies neutralized MPXV clades Ib and IIb and vaccinia virus (VACV) and cross-reacted with OPG153 orthologs from cowpox and variola viruses. Immunization with MPXV OPG153 elicited a potent neutralizing antibody response against MPXV and VACV in mice, substantiating OPG153 as both a promising vaccine antigen and a potent target for preventive and therapeutic antibodies.

An unprecedented outbreak of Oropouche virus (OROV) is occurring in the Americas, characterized by thousands of confirmed cases and a wide geographical spread, including areas outside the Amazon Basin. Little is known about this neglected arbovirus regarding its pathophysiological aspects and potentially different transmission modes. This study describes the clinical course of a man who returned from a trip to Cuba and presented to our hospital 4 days after the onset of febrile symptoms. The patient was diagnosed with Oropouche fever and was followed for 177 days after the onset of symptoms. We performed a longitudinal investigation of the samples collected from several body sites (whole blood, serum, urine, and semen) with the aim of providing further insights into OROV infection dynamics, using the detection of antigenomic RNA as a marker of active viral replication. Clinical samples that were longitudinally collected over the course of OROV infection showed consistently higher amounts of antigenomic RNA compared to genomic RNA, even after viral clearance from serum. Moreover, our case study showed the persistence of OROV RNA in serum of less than 15 days from the onset of symptoms, as compared to up to one month in urine, three months in semen, and four months in whole blood. Our study suggests that Oropouche virus may persist in an actively replicating state in different body sites for long periods of time, with important implications for transmission dynamics. Furthermore, our results provide a diagnostic indication, suggesting that serum is inferior to both urine and whole blood as preferred diagnostic samples. Further studies are needed to determine the pathogenetic implications of these findings, as they have been derived from a single case and must be confirmed using a larger number of cases.

Background: Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV. Methods: The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs). Results: The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR. Conclusion: We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs.

Acute gastroenteritis is a common condition with a wide and often indistinguishable etiology, requiring laboratory support for an accurate diagnosis. Classical diagnostic methods, including stool culture and antigen-based tests, are limited by restricted pathogen coverage and variable sensitivity. In the present study, 548 stool samples from patients with suspected gastroenteritis were tested using the QIAstat-Dx Gastrointestinal Panel 2 and compared with stool culture and rotavirus/adenovirus antigen tests. The molecular panel showed a positivity rate of 50.4%, consistently higher than stool culture (12.6%) and antigen assays (12.3% for rotavirus and 4.4% for adenovirus). The most frequently detected pathogens included enteropathogenic Escherichia coli (15.3%), Campylobacter spp. (12.0%), and enteroaggregative E. coli (10.2%). Agreement between methods was good for bacterial pathogens but low for viral targets. Discordant viral results were often associated with low antigen cut-off index values and multiple pathogen detections by the molecular panel, suggesting potential limitations of antigen-based assays. Overall, the QIAstat-Dx Gastrointestinal Panel 2 improves pathogen detection and provides rapid, comprehensive diagnostic information, while a combined approach with conventional methods may represent the most appropriate strategy for optimizing patient management.

Oropouche virus (OROV) has re-emerged as a significant public health threat during the 2023–2025 outbreak, characterized by an unprecedented geographic expansion beyond endemic regions. The virus has been detected in various regions, including the Caribbean, as well as in Europe and the United States, among travelers from Central and South America. This epidemic was associated with the expansion of a novel reassortant strain, which may exhibit enhanced viral fitness and immune evasion properties. Although orthobunyaviruses are known to counteract interferon responses, the specific strategy adopted by OROV remains incompletely understood. In this study, we compared the in-vitro behavior of the prototype strain BeAn19991 and a 2024 isolate representing the reassortant virus responsible for the 2023–2025 epidemic (ISCDC-2024), focusing on innate immune response. We examined the sensitivity of the two strains to IFN-β antiviral activity and their ability to induce type I IFN in fibroblast. Furthermore, we characterized the innate immune response triggered by in-vitro infection of PBMCs from healthy donors. Although the two viral strains exhibited a similar plateau in viral RNA accumulation and comparable sensitivity to exogenous IFN-β, the IFN-β induction in fibroblasts was delayed, with higher levels induced by ISCDC-2024 than by BeAn19991. Moreover, the two strains displayed distinct innate immune profiles in PBMCs: BeAn19991 predominantly induced a robust antiviral IFN-driven response, whereas ISCDC-2024 infection was associated with an IL-6-dominated inflammatory pattern. Overall, these findings highlight distinct phenotypic features of the contemporary reassortant isolate and suggest strain-specific differences in the modulation of innate immunity, potentially reflecting distinct immune evasion strategies. If confirmed, this hypothesis could have significant implications for infection transmission, viral persistence and disease severity, highlighting the need for continuous surveillance and functional characterization of emerging OROV strains to drive public health interventions and antiviral countermeasures.