Background: The Oropouche virus (OROV) is an arthropod-borne virus that causes an acute febrile illness similar to other arboviral diseases. In 2024, Oropouche cases sharply increased in several countries of the Americas, including Bolivia. Here, we performed a genomic study to investigate the origin and spread of OROV in the Bolivian Amazon region. Methods: Full-length OROV genomes from 34 positive samples collected in the three affected Bolivian departments during 2024 were sequenced using an amplicon-based approach. Maximum Likelihood (ML) phylogenetic analyses of separate viral segments were conducted to identify the responsible viral lineage. Bayesian phylogeographic analysis of concatenated viral segments was used to reconstruct the viral spatiotemporal dispersion pattern within the country. Findings: Epidemiological data shows that the first OROV-positive cases in 2024 in Bolivia were reported in samples collected from the Pando department during mid-January, and the peak of OROV-positive cases occurred in mid-April. The phylogenetic analysis of OROV genomes revealed that all cases detected in Bolivia belong to the novel reassortant OROV clade that drove the recent epidemic in Brazil. Our phylogeographic analysis detected at least two exportation events of OROV from the Brazilian state of Acre to the Bolivian municipalities of Guayaramerin and Riberalta, both located in the Beni department, with subsequent dissemination to municipalities of Pando and La Paz departments. Viral introductions probably occurred between early October and early November 2023, thus indicating a lag of about three months between OROV introduction and detection in Bolivia. Interpretation: Our findings confirm that OROV spread at least twice from the western Brazilian Amazon to the neighboring Bolivian department of Beni in late 2023, successfully establishing regional transmission chains. The introduction and spread of OROV in Bolivia coincides with the Amazonian rainy season, from November to April, a period likely marked by an increase in vector abundance. These findings underscore the critical need for active OROV surveillance across the cross-border Amazonian region between Brazil and Bolivia. They also confirm the potential for sustained OROV transmission within the Bolivian Amazon, highlighting the importance of preparedness for future outbreaks.

The authors have declared no competing interest.

This publication was in part supported by the Cooperative Agreement Number NU50CK000639 awarded to the Pan American Health Organization and funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics Research Committee of the Universidad Cristiana de Bolivia (FWA00028929) approved this study under protocol number CEI/001/2024.

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Yes

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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All OROV consensus sequences generated in this study (n = 102, corresponding to the three segments of 34 unique samples) were deposited in GenBank (https://www.ncbi.nml.nih.gov/ genbank) under the accession numbers PQ634468 - PQ634569.

https://www.ncbi.nml.nih.gov/genbank