Countrywide Spread and Spatiotemporal Diffusion Dynamics of Oropouche virus in Cuba, 2024

Countrywide Spread and Spatiotemporal Diffusion Dynamics of Oropouche virus in Cuba, 2024 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Countrywide Spread and Spatiotemporal Diffusion Dynamics of Oropouche virus in Cuba, 2024 Maria Guzman, Rosario Gravier, Melissa Perez, Mayling Alvarez, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7303560/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Oropouche virus (OROV) was reported in Cuba in May 2024 and rapidly spread throughout the country. Among 147 RT-PCR-positive cases identified from May to July, we generated 39 whole-genome sequences. Phylogenetic analysis revealed that all sequences formed a monophyletic cluster nested within the novel reassortant OROVBR-2015-2025, which has been circulating extensively in Brazil since the end of 2023. Additional analyses demonstrated that the Cuban sub-clade originated from a single viral introduction from the Brazilian state of Acre, probably in early February 2024, and circulated cryptically until its identification in May. The introduction likely occurred in the Central region of the country, from which the virus spread and established secondary transmission hubs in the Western and Eastern regions. These findings underscore the capacity of OROV to spread well beyond the Amazon region, which was considered its endemic area of circulation. Biological sciences/Evolution/Evolutionary genetics Health sciences/Medical research/Epidemiology Figures Figure 1 Figure 2 Figure 3 MAIN Oropouche virus (OROV) was first identified in Trinidad and Tobago during the 1950s and later in South and Central America 1 . OROV generally causes a mild illness although more severe manifestations like meningitis, encephalitis, Guillain-Barré syndrome, and recently vertical transmission, have also been reported 2,3 . OROV has a single-stranded, negative-sense RNA genome divided into three segments, designated according to their size as L (large, 6.85 kb), M (medium, 4.36 kb), and S (small, 0.95 kb) 1 . OROV is transmitted between mammals mainly through the bite of infected Culicoides paraensis midges, although Culex quinquefasciatus has been described as a potential secondary vector in urban settings 1,4,5 . Since 2024, OROV has spread beyond the Amazon region in Brazil, prompting the Pan American Health Organization (PAHO) to issue an alert regarding its circulation in the Americas. 6 On May 27, 2024, the Cuban Ministry of Public Health confirmed the first detection of local OROV transmission in the country. 7 Acute Febrile Illness (AFI) surveillance in Santiago de Cuba province, identified a surge of cases that tested negative for dengue virus. Soon, the Arbovirus National Reference Laboratory at the Institute of Tropical Medicine “Pedro Kouri” identified OROV as the cause of the outbreak. By August 28, 506 confirmed cases had been reported across 99 out of 168 municipalities in all of the country’s 7 . Recent studies demonstrated that the ongoing OROV outbreak in Brazil resulted from the sustained transmission and dissemination of a novel OROV reassortant lineage, which likely emerged in the Amazonas state between 2010 and 2014 8 and recently spread to non-endemic regions of the country in 2024 9 . Preliminary phylogenetic analyses, on the S segment of Cuban samples of Santiago de Cuba and Cienfuegos provinces, suggested that the Cuban sequences were closely related to 2023 OROV sequences from Brazil 10 . To better understand the origin and temporality of the first-ever reported OROV introduction in Cuba, as well as the dynamics of its rapid spread throughout the country, we sequencing and analyzing full-length OROV genomes from serum samples collected from Oropouche-confirmed patients between May 12 and July 9, 2024, in 14 of 16 first-level administrative divisions. In this period, samples from 217 suspected Oropouche cases were processed by real-time RT-PCR 11 with 147 (67.8%) tested positive for OROV mRNA. We selected 39 samples (26.5% of positive cases) for whole-genome sequencing based on Ct values (≤28.5) and geographical and temporal representativeness ( Supplementary Fig. 1 ). Maximum likelihood (ML) phylogenetic analyses performed individually for each genomic segment revealed that all OROV sequences belong to the novel reassortant clade (OROV BR-2015-2024 ) detected in Brazil 8 ( Fig- 1 ). To resolve the phylogenetic relationship between Cuban and Brazilian OROV sequences with more resolution, we conducted an ML phylogenetic analysis on concatenated L, M, and S segments and the dataset used previously 8 . This analysis showed that, within the OROVBR-2015-2024 clade, Cuban OROV sequences formed a strongly supported (aLRT = 1) monophyletic cluster (OROV-CU), closely related to the Brazilian sub-clades AMACRO-I and AMACRO-II ( Fig 2a ). These sub-clades comprise sequences from Brazilian states distributed across Northern (Amazonas, Acre, and Roraima), Southeastern (Rio de Janeiro and Espírito Santo), and Southern (Paraná and Santa Catarina) country regions 8 . Thus, we performed a Discrete Bayesian phylogeographic analysis with concatenated segments to model the viral diffusion process between Brazil and Cuba. For this analysis, we retained the oldest sequence of OROV BR-2015-2024 clade, detected in the Amazonas state in 2015, all OROV sequences belonging to AMACRO-I and AMACRO-II (2023-2024) sub-clades, and the Cuban sequences of this study. This analysis showed that the OROV-CU sub-clade most probably originated from a single introduction event from the Brazilian state of Acre [ PSP = 1.0] around early February 2024 (T MRCA = 2024-02-10 [95% HPD: 2024-01-04 - 2024-03-17]) ( Fig. 2b ). Supplementary figure 2 shows the international dissemination of the Cuban OROV clade 12 . We then use geographical coordinates of patient residential area to reconstructed the fine-scale dispersion of the OROV-CU clade through a continuous spatial diffusion model with non-homogeneous dispersion rates ( Fig 3a ). Our analysis suggests that Central Cuba—Ciego de Ávila, Sancti Spíritus, and Camagüey provinces — served as the virus entry point in the country being the initial epicenter of its spread. An eastern hub emerged in Mayabeque and Artemisa provinces disseminating to westward to Pinar del Río province and eastward to Matanzas province. At the same time, in the Eastern region, the virus expanded from Holguín province to Santiago de Cuba and Guantánamo provinces. By late July 2024, the Central Cuba epicenter seeded additional infections in the Western provinces of La Habana and Pinar del Río. Continuous phylogeographic reconstructions also revealed that the median distance of viral migrations was 22 km (standard deviation: 91 km; interquartile range: 39 km), remaining relatively constant throughout the study period ( Fig. 3b ). Our analysis also demonstrated that very-short-distance migrations ( 30 km, 37%) ( Fig. 3c ). We estimated the OROV median dispersion velocity within Cuba during the study period of 1.90 km/day (95% HPD: 0.77 – 3.18 km/day), remaining relatively stable over the study period ( Fig. 3d ). Our results confirm that the Cuban outbreak is linked to the spread of viruses belonging to the OROVBR-2015-2025 clade, which actively circulates in Brazil. All Cuban sequences were part of a monophyletic cluster (OROV-CU) nested within the AMACRO-II subclade of OROVBR-2015-2025, which also comprises all OROV sequences detected in the Brazilian states of Acre, Rondônia, and the southern parts of the Amazonas states from December 2023 onwards 8 . OROV sequences from cases imported from Cuba in Europe 12 also clustered with the OROV-CU sub-clade. Our phylogeographic analysis further suggests that the Cuban outbreak resulted from a single virus introduction, likely occurring in February 2024, originating from the Brazilian state of Acre. This would imply that the virus underwent a period of silent transmission for approximately three months before being detected at the end of May. Intense travel between Cuba and Brazil, along with the high circulation of the AMACRO-II subclade in Brazil early 2024, likely facilitated the introduction of OROV into Cuba. Although the OROV transmission was recognized at the same time (end of May), in eastern (Santiago de Cuba), central (Cienfuegos and Villa Clara), and western (Mayabeque and Matanzas) provinces, 10 ; our phylogeographic results suggest that the initial introduction of the virus occurred in the Central provinces. From there, OROV spread concomitantly to the eastern and western provinces. Previous studies of the OROV BR-2015-2025 clade in Brazil, estimated that its average dispersion rate was 1.00 km/day in Acre, Rondônia, Roraima states and the southern parts of the Amazonas state (AMACRO region) and 0.66 km/day in the rest of the Amazonas state with most dispersal events being consistent with the typical flight range of Culicoides vectors 8 . However, the Cuban subclade exhibited a stable dispersal velocity of 1.90 km/day (95% HPD: 0.77–3.18 km/day), almost twice that observed in Roraima state and AMACRO, and nearly three times that observed in Amazonas state, Brazil. Similarly, continuous phylogeographic reconstructions in Cuba showed that very-short (30 km) distance spread represented 8%, 22%, 33%, and 37% of the viral movements, respectively. At the same time, these proportions were 65%, 5%, 9%, and 22% in Brazil. This increased speed of transmission could be due to factors such as the virus circulation into new environments, the naïve population, population density, and intensive human mobility among the Cuban provinces. Differences in vectors' behavior might also contribute to this difference. We have recently detected OROV in pools of Culex quinquefasciatus mosquitoes and of Ceratopogonidae spp 10 . Additionally, the presence of Culicoides paraensis , was confirmed in the country 13 . More studies are needed to better define others vector implications the transmission in Cuba considering that it has occurred in semi-urban, urban, and rural environments. Our study has some limitations a) although we selected samples to ensure geographical and temporal representativeness, only 26.5% of the cases identified during the study period were sequenced b) samples with higher RT-PCR Ct values were excluded due to the low likelihood of sequencing success c) we did not investigate negative dengue samples collected before the recognition of OROV transmission in Cuba. The OROV autochthonous transmission in Cuba demonstrates the virus capacity to spread beyond Amazon areas traditionally considered as endemic. This raises concerns about the potential involvement of unrecognized vectors. Given that environmental, climatic, and demographic changes are global phenomena, it is not surprising that OROV could spread further across the Americas and beyond, being crucial to strengthen global efforts in enhancing surveillance, improving vector control strategies, and advancing research into the factors driving virus transmission Declarations Data availability All the OROV genomes generated in this study were deposited at GISAID under accession numbers EPI_ISL_19611792 - EPI_ISL_19611830 (https://doi.org/10.55876/gis8.241214tm). Sample data can be found in Supplementary Table . Acknowledgements This study is supported by the Cuban Ministry of Health¸ FAPEAM Call 04/2022/FIOCRUZ/FAPEAM/FAPERO - INOVAÇÃO NA AMAZÔNIA: F.G.N.; FAPEAM Call 023/2022 - INICIATIVA AMAZÔNIA + 10: F.G.N. Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq Institutos Nacionais de Ciência e Tecnologia (INCT - VER) e Chamada CNPq/MCTI 10/2023 - Faixa B - Grupos Consolidados - Universal 2023 (421620/2023-4) and the Pan American Health Organization. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Author contribution MGG and FGN contributed to study conception and study design. RG, MMP, MA, LP, AJB, DH, SS, VS, MM, GB, IA, FGN, MGG contributed to laboratory work. RG, MMP, MA, LP, AJB, DH, SS, JRA, CP, MR, LF, LG, VS, MM, GB, IA, SR, VK, FGN, MGG contributed to data analysis. RG, VS, GB, IA, FGN contributed to results visualization. GB, IA, FGN, MGG contributed to write the original manuscript. RG, MMP, MA, LP, AJB, DH, SS, JRA, CP, MR, LF, JL, LG, VS, MM, GB, IA, SR, VK, FGN, MGG contributed to manuscript revision. MGG, FGN, LF, LG, JMR resources contribution. References Sakkas, H., Bozidis, P., Franks, A. & Papadopoulou, C. Oropouche Fever: A Review. Viruses 10 (2018). de Armas Fernández, J.R. , et al. Report of an unusual association of Oropouche Fever with Guillain-Barré syndrome in Cuba, 2024. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 43 , 2233-2237 (2024). PAHO. Oropouche: Cases of mother-to-child transmission under investigation in Brazil. (2024). Travassos da Rosa, J.F. , et al. Oropouche Virus: Clinical, Epidemiological, and Molecular Aspects of a Neglected Orthobunyavirus. The American journal of tropical medicine and hygiene 96 , 1019-1030 (2017). Feitoza, L.H.M. , et al. Influence of meteorological and seasonal parameters on the activity of Culicoides paraensis (Diptera: Ceratopogonidae), an annoying anthropophilic biting midge and putative vector of Oropouche Virus in Rondônia, Brazilian Amazon. Acta tropica 243 , 106928 (2023). PAHO. Epidemiological Alert - Oropouche in the Americas Region - 13 December 2024. (2025). MINSAP. Nota informativa del Ministerio de Salud Pública 27 de mayo de 2024. (2024). Naveca, F.G. , et al. Human outbreaks of a novel reassortant Oropouche virus in the Brazilian Amazon region. Nature medicine (2024). Gräf, T. , et al. Long-Range Spread and Sustained Transmission of Oropouche Virus Outside the Endemic Brazilian Amazon Region. The Lancet. Infectious diseases (2024). Benitez, A.J. , et al. Oropouche Fever, Cuba, May 2024. Emerging infectious diseases 30 (2024). Naveca, F.G. , et al. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses. Memorias do Instituto Oswaldo Cruz 112 , 510-513 (2017). Deiana, M. , et al. Full Genome Characterization of the First Oropouche Virus Isolate Imported in Europe from Cuba. Viruses 16 (2024). Pérez, Y.M. , et al. First report of Culicoides paraensis (Goeldi, 1905) (Diptera: Ceratoponidae) in Cuba: A new challenge for public health. Parasite epidemiology and control 29 , e00423 (2025). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarytable.xlsx Anonymized patient information data SupplementaryFigure1.docx Oropouche cases in Cuba and OROV genomes by epidemiological week. The blue bars represent the number of Oropouche cases throughout the country, whereas the green bars represent the number of genomes g SupplementaryFigure2.docx International Dissemination of the Cuban OROV Clade. Maximum likelihood phylogenetic tree based on concatenated OROV genomes (n = 284) from the OROVBR-2015-2024 clade, including Cuban OROV genomes (n BC144298RS.pdf Reporting Summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7303560","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":500647581,"identity":"cb265b9b-5ae0-4539-800b-ebed28bd065d","order_by":0,"name":"Maria 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1","display":"","copyAsset":false,"role":"figure","size":57286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum likelihood (ML) phylogenetic analyses of the M, L, and S segments of Cuban OROV sequences\u003c/strong\u003e. Phylogenetic trees were constructed based on the M (a), L (b), and S (c) segments of 131 complete OROV genomes, including the 39 genomes generated in this study and 92 prototypical sequences representing other OROV lineages from different countries and years, as suggested by Naveca \u003cem\u003eet. al.\u003c/em\u003e 2024\u003csup\u003e8\u003c/sup\u003e. Brackets denote major OROV clades, each labeled with their respective designations and statistical support (SH-aLRT 1,000 replicates). Trees are scaled according to the genetic distance. Cuban and prototypical OROV sequences are represented in colors following the coding scheme shown in the left panel and their genotypical classification.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/c7cb21e5bf1d361c8bfe6dce.png"},{"id":89386342,"identity":"76611cb6-3332-4079-9c61-b9d314d788eb","added_by":"auto","created_at":"2025-08-19 12:35:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal dynamics of OROV-CU clade emergence.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Maximum likelihood phylogenetic tree based on concatenated OROV genomes (n = 282) from the OROVBR-2015-2024 clade, including Cuban OROV genomes (n = 39). Clusters are annotated \u003csup\u003e8\u003c/sup\u003e and statistical support values (aLRT) shown. The tree is scaled according to the legend at the bottom of the panel. \u003cstrong\u003eb.\u003c/strong\u003e Bayesian time-scaled Maximum Clade Credibility (MCC) tree (n = 117) for the clusters highlighted by a dashed line in the previous panel. This subset includes sequences from the Cuban clade and Brazilian clades AMACRO-I (n = 34) and AMACRO-II (n = 43), covering data from the northern Brazilian states of Amazonas, Acre, and Roraima, southeastern states of Rio de Janeiro and Espírito Santo, and southern states of Paraná and Santa Catarina. Branches in the MCC tree are color-coded according to the inferred ancestral location of their basal nodes, as indicated by the legend on the right. The posterior statistical of the OROV-CU clade is annotated, while an Amazonas sequence from 2015 was not shown for clarity. \u003cstrong\u003ec\u003c/strong\u003e. The map mirrors tree colors and marks Cuba and relevant Brazilian states associated with the Cuban clade emergence.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/316af1f1d1c1d2372bad98cd.png"},{"id":89388796,"identity":"ef519eaf-0a26-4bf3-b847-6f9e32f98441","added_by":"auto","created_at":"2025-08-19 12:43:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":149680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal dynamics of the OROV-CU clade\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e. Dissemination of the OROV-CU clade (n = 39) across Cuba at the provincial level. Lines indicate viral migration, with colors corresponding to the time of occurrence. \u003cstrong\u003eb\u003c/strong\u003e. Distance of viral migrations during the first half of 2024. Data points are colored according to the time of occurrence. The median distance of viral migrations is shown (dotted line). \u003cstrong\u003ec\u003c/strong\u003e. Frequency distribution of short (\u0026lt; 5km), medium (5 – 30 km), and long (\u0026gt; 30 km) distance viral migrations calculated from the branches of 1,000 randomly selected trees from the posterior distribution of the continuous phylogeographic analysis. \u003cstrong\u003ed\u003c/strong\u003e. Weighted branch dispersal velocity of the OROV-CU clade through time (posterior median = solid line, 95% HPD = pale areas).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/6931db6bab1845867cab116b.png"},{"id":89389871,"identity":"0c8cc5d3-a047-4b1d-b653-8739ae979cea","added_by":"auto","created_at":"2025-08-19 12:51:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":820011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/4c7aa415-2a40-459a-a3f9-2d0923560e06.pdf"},{"id":89386343,"identity":"114e5e73-7786-4fee-901d-8f395219b6f1","added_by":"auto","created_at":"2025-08-19 12:35:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11304,"visible":true,"origin":"","legend":"Anonymized patient information data","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/47810e3ecc75c1c5c091c882.xlsx"},{"id":89389870,"identity":"cc9ee8c8-e2ec-4e7b-900e-674d72aaf325","added_by":"auto","created_at":"2025-08-19 12:51:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":183218,"visible":true,"origin":"","legend":"Oropouche cases in Cuba and OROV genomes by epidemiological week. The blue bars represent the number of Oropouche cases throughout the country, whereas the green bars represent the number of genomes g","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/6e1331562992c02b96a94fb3.docx"},{"id":89386344,"identity":"06d663cb-485a-41d4-b72a-77c54033cc6a","added_by":"auto","created_at":"2025-08-19 12:35:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":176306,"visible":true,"origin":"","legend":"International Dissemination of the Cuban OROV Clade. Maximum likelihood phylogenetic tree based on concatenated OROV genomes (n = 284) from the OROVBR-2015-2024 clade, including Cuban OROV genomes (n","description":"","filename":"SupplementaryFigure2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/77c2df6c47420f5ae2673a71.docx"},{"id":89386347,"identity":"449c7faf-c13b-4e61-9602-cd6f93236ab5","added_by":"auto","created_at":"2025-08-19 12:35:32","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1777947,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"BC144298RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7303560/v1/c5ca32cc00782f46337aa444.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Countrywide Spread and Spatiotemporal Diffusion Dynamics of Oropouche virus in Cuba, 2024","fulltext":[{"header":"MAIN","content":"\u003cp\u003eOropouche virus (OROV) was first identified in Trinidad and Tobago during the 1950s and later in South and Central America \u003csup\u003e1\u003c/sup\u003e. OROV generally causes a mild illness although more severe manifestations like meningitis, encephalitis, Guillain-Barr\u0026eacute; syndrome, and recently vertical transmission, have also been reported\u003csup\u003e2,3\u003c/sup\u003e. OROV has a single-stranded, negative-sense RNA genome divided into three segments, designated according to their size as L (large, 6.85 kb), M (medium, 4.36 kb), and S (small, 0.95 kb) \u003csup\u003e1\u003c/sup\u003e. OROV is transmitted between mammals mainly through the bite of infected \u003cem\u003eCulicoides paraensis\u003c/em\u003e midges, although \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e has been described as a potential secondary vector in urban settings\u003csup\u003e1,4,5\u003c/sup\u003e. Since 2024, OROV has spread beyond the Amazon region in Brazil, prompting the Pan American Health Organization (PAHO) to issue an alert regarding its circulation in the Americas.\u0026nbsp;\u003csup\u003e6\u003c/sup\u003e On May 27, 2024, the Cuban Ministry of Public Health confirmed the first detection of local OROV transmission in the country.\u003csup\u003e7\u003c/sup\u003e Acute Febrile Illness (AFI) surveillance in Santiago de Cuba province, identified a surge of cases that tested negative for dengue virus.\u0026nbsp; Soon, the Arbovirus National Reference Laboratory at the Institute of Tropical Medicine \u0026ldquo;Pedro Kouri\u0026rdquo; identified OROV as the cause of the outbreak. By August 28, 506 confirmed cases had been reported across 99 out of 168 municipalities in all of the country\u0026rsquo;s\u0026nbsp;\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRecent studies demonstrated that the ongoing OROV outbreak in Brazil resulted from the sustained transmission and dissemination of a novel OROV reassortant lineage, which likely emerged in the Amazonas state between 2010 and 2014 \u003csup\u003e8\u003c/sup\u003e and recently spread to non-endemic regions of the country in 2024 \u003csup\u003e9\u003c/sup\u003e. Preliminary phylogenetic analyses, on the S segment of Cuban samples of Santiago de Cuba and Cienfuegos provinces, suggested that the Cuban sequences were closely related to 2023 OROV sequences from Brazil \u003csup\u003e10\u003c/sup\u003e. To better understand the origin and temporality of the first-ever reported OROV introduction in Cuba, as well as the dynamics of its rapid spread throughout the country, \u0026nbsp;we sequencing and analyzing full-length OROV genomes from \u0026nbsp;serum samples collected from Oropouche-confirmed patients between May 12 and July 9, 2024, in 14 of 16 first-level administrative divisions.\u003c/p\u003e\n\u003cp\u003eIn this period, samples from 217 suspected Oropouche cases were processed by real-time RT-PCR \u003csup\u003e11\u003c/sup\u003e with 147 (67.8%) tested positive for OROV mRNA. We selected 39 samples (26.5% of positive cases) for whole-genome sequencing based on Ct values (\u0026le;28.5) and geographical and temporal representativeness (\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eFig. 1\u003c/strong\u003e). Maximum likelihood (ML) phylogenetic analyses performed individually for each genomic segment revealed that all OROV sequences belong to the novel reassortant clade (OROV\u003csub\u003eBR-2015-2024\u003c/sub\u003e) detected in Brazil \u003csup\u003e8\u003c/sup\u003e (\u003cstrong\u003eFig- 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo resolve the phylogenetic relationship between Cuban and Brazilian OROV sequences with more resolution, we conducted an ML phylogenetic analysis on concatenated L, M, and S segments and the dataset used previously\u003csup\u003e8\u003c/sup\u003e. This analysis showed that, within the OROVBR-2015-2024 clade, Cuban OROV sequences formed a strongly supported (aLRT = 1) monophyletic cluster (OROV-CU), closely related to the Brazilian sub-clades AMACRO-I and AMACRO-II (\u003cstrong\u003eFig 2a\u003c/strong\u003e). These sub-clades comprise sequences from Brazilian states distributed across Northern (Amazonas, Acre, and Roraima), Southeastern (Rio de Janeiro and Esp\u0026iacute;rito Santo), and Southern (Paran\u0026aacute; and Santa Catarina) country regions \u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThus, we performed a Discrete Bayesian phylogeographic analysis with concatenated segments to model the viral diffusion process between Brazil and Cuba. For this analysis, we retained the oldest sequence of OROV\u003csub\u003eBR-2015-2024\u003c/sub\u003e clade, detected in the Amazonas state in 2015, all OROV sequences belonging to AMACRO-I and AMACRO-II (2023-2024) sub-clades, and the Cuban sequences of this study. This analysis showed that the OROV-CU sub-clade most probably originated from a single introduction event from the Brazilian state of Acre [\u003cem\u003ePSP\u003c/em\u003e = 1.0] around early February 2024 (T\u003csub\u003eMRCA\u003c/sub\u003e = 2024-02-10 [95% HPD: 2024-01-04 - 2024-03-17]) (\u003cstrong\u003eFig. 2b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary figure 2\u003c/strong\u003e shows the international dissemination of the Cuban OROV clade\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe then use geographical coordinates of patient residential area to reconstructed the fine-scale dispersion of the OROV-CU clade through a continuous spatial diffusion model with non-homogeneous dispersion rates (\u003cstrong\u003eFig 3a\u003c/strong\u003e). \u0026nbsp;Our analysis suggests that Central Cuba\u0026mdash;Ciego de \u0026Aacute;vila, Sancti Sp\u0026iacute;ritus, and Camag\u0026uuml;ey provinces \u0026mdash; served as the virus entry point in the country being the initial epicenter of its spread. An eastern hub emerged in Mayabeque and Artemisa provinces disseminating to westward to Pinar del R\u0026iacute;o province and eastward to Matanzas province. At the same time, in the Eastern region, the virus expanded from Holgu\u0026iacute;n province to Santiago de Cuba and Guant\u0026aacute;namo provinces. By late July 2024, the Central Cuba epicenter seeded additional infections in the Western provinces of La Habana and Pinar del R\u0026iacute;o.\u003c/p\u003e\n\u003cp\u003eContinuous phylogeographic reconstructions also revealed that the median distance of viral migrations was 22 km (standard deviation: 91 km; interquartile range: 39 km), remaining relatively constant throughout the study period (\u003cstrong\u003eFig. 3b\u003c/strong\u003e). Our analysis also demonstrated that very-short-distance migrations (\u0026lt;2 km) constituted 8% of total viral movements, followed by short (2\u0026ndash;10 km, 22%), medium (11\u0026ndash;30 km, 33%), and long-distance migrations (\u0026gt; 30 km, 37%) (\u003cstrong\u003eFig. 3c\u003c/strong\u003e). We estimated the OROV median dispersion velocity within Cuba during the study period of 1.90 km/day (95% HPD: 0.77 \u0026ndash; 3.18 km/day), remaining relatively stable over the study period (\u003cstrong\u003eFig. 3d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eOur results confirm that the Cuban outbreak is linked to the spread of viruses belonging to the OROVBR-2015-2025 clade, which actively circulates in Brazil. All Cuban sequences were part of a monophyletic cluster (OROV-CU) nested within the AMACRO-II subclade of OROVBR-2015-2025, which also comprises all OROV sequences detected in the Brazilian states of Acre, Rond\u0026ocirc;nia, and the southern parts of the Amazonas states from December 2023 onwards \u003csup\u003e8\u003c/sup\u003e. OROV sequences from cases imported from Cuba in Europe \u003csup\u003e12\u003c/sup\u003e also clustered with the OROV-CU sub-clade.\u003c/p\u003e\n\u003cp skip=\"true\"\u003eOur phylogeographic analysis further suggests that the Cuban outbreak resulted from a single virus introduction, likely occurring in February 2024, originating from the Brazilian state of Acre. This would imply that the virus underwent a period of silent transmission for approximately three months before being detected at the end of May. Intense travel between Cuba and Brazil, along with the high circulation of the AMACRO-II subclade in Brazil early 2024, likely facilitated the introduction of OROV into Cuba.\u003c/p\u003e\n\u003cp\u003eAlthough the OROV transmission was recognized at the same time (end of May), in eastern (Santiago de Cuba), central (Cienfuegos and Villa Clara), and western (Mayabeque and Matanzas) provinces, \u003csup\u003e10\u003c/sup\u003e; our phylogeographic results suggest that the initial introduction of the virus occurred in the Central provinces. From there, OROV spread concomitantly to the eastern and western provinces.\u003c/p\u003e\n\u003cp\u003ePrevious studies of the OROV\u003csub\u003eBR-2015-2025\u003c/sub\u003e clade in Brazil, estimated that its average dispersion rate was 1.00 km/day in Acre, Rond\u0026ocirc;nia, Roraima states and the southern parts of the Amazonas state (AMACRO region) and 0.66 km/day in the rest of the Amazonas state with most dispersal events being consistent with the typical flight range of Culicoides vectors \u003csup\u003e8\u003c/sup\u003e. However, the Cuban subclade exhibited a stable dispersal velocity of 1.90 km/day (95% HPD: 0.77\u0026ndash;3.18 km/day), almost twice that observed in Roraima state and AMACRO, and nearly three times that observed in Amazonas state, Brazil. Similarly, continuous phylogeographic reconstructions in Cuba showed that very-short (\u0026lt; 2 km), short (2\u0026ndash;10 km), medium (11\u0026ndash;30 km), and long (\u0026gt;30 km) distance spread represented 8%, 22%, 33%, and 37% of the viral movements, respectively. At the same time, these proportions were 65%, 5%, 9%, and 22% in Brazil. This increased speed of transmission could be due to factors such as the virus circulation into new environments, the na\u0026iuml;ve population, population density, and intensive human mobility among the Cuban provinces. Differences in vectors\u0026apos; behavior might also contribute to this difference. We have recently detected OROV in pools of \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e mosquitoes and of\u0026nbsp;\u003cem\u003eCeratopogonidae\u003c/em\u003e spp\u0026nbsp;\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e. Additionally, the presence of \u003cem\u003eCulicoides paraensis\u003c/em\u003e, was confirmed in the country\u003csup\u003e13\u003c/sup\u003e. More studies are needed to better define others vector implications the transmission in Cuba considering that it has occurred in semi-urban, urban, and rural environments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study has some limitations a) although we selected samples to ensure geographical and temporal representativeness, only 26.5% of the cases identified during the study period were sequenced b) samples with higher RT-PCR Ct values were excluded due to the low likelihood of sequencing success c) we did not investigate negative dengue samples collected before the recognition of OROV transmission in Cuba.\u003c/p\u003e\n\u003cp\u003eThe OROV autochthonous transmission in Cuba demonstrates the virus capacity to spread beyond Amazon areas traditionally considered as endemic. This raises concerns about the potential involvement of unrecognized vectors. Given that environmental, climatic, and demographic changes are global phenomena, it is not surprising that OROV could spread further across the Americas and beyond, being crucial to strengthen global efforts in enhancing surveillance, improving vector control strategies, and advancing research into the factors driving virus transmission\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll the OROV genomes generated in this study were deposited at GISAID under accession numbers EPI_ISL_19611792 - EPI_ISL_19611830 (https://doi.org/10.55876/gis8.241214tm). Sample data can be found in \u003cstrong\u003eSupplementary \u0026nbsp;Table\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the Cuban Ministry of Health¸ FAPEAM Call 04/2022/FIOCRUZ/FAPEAM/FAPERO - INOVAÇÃO NA AMAZÔNIA: F.G.N.; FAPEAM Call 023/2022 - INICIATIVA AMAZÔNIA + 10: F.G.N. Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq Institutos Nacionais de Ciência e Tecnologia (INCT - VER) e Chamada CNPq/MCTI 10/2023 - Faixa B - Grupos Consolidados - Universal 2023 (421620/2023-4) and the Pan American Health Organization. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMGG and FGN contributed to study conception and study design. RG, MMP, MA, LP, AJB, DH, SS, VS, MM, GB, IA, FGN, MGG contributed to laboratory work. RG, MMP, MA, LP, AJB, DH, SS, JRA, CP, MR, LF, LG, VS, MM, GB, IA, SR, VK, FGN, MGG contributed to data analysis. RG, VS, GB, IA, FGN contributed to results visualization. GB, IA, FGN, MGG contributed to write the original manuscript. RG, MMP, MA, LP, AJB, DH, SS, JRA, CP, MR, LF, JL, LG, VS, MM, GB, IA, SR, VK, FGN, MGG contributed to manuscript revision. MGG, FGN, LF, LG, JMR resources contribution.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSakkas, H., Bozidis, P., Franks, A. \u0026amp; Papadopoulou, C. Oropouche Fever: A Review. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(2018).\u003c/li\u003e\n\u003cli\u003ede Armas Fern\u0026aacute;ndez, J.R.\u003cem\u003e, et al.\u003c/em\u003e Report of an unusual association of Oropouche Fever with Guillain-Barr\u0026eacute; syndrome in Cuba, 2024. \u003cem\u003eEuropean journal of clinical microbiology \u0026amp; infectious diseases : official publication of the European Society of Clinical Microbiology\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 2233-2237 (2024).\u003c/li\u003e\n\u003cli\u003ePAHO. Oropouche: Cases of mother-to-child transmission under investigation in Brazil. (2024).\u003c/li\u003e\n\u003cli\u003eTravassos da Rosa, J.F.\u003cem\u003e, et al.\u003c/em\u003e Oropouche Virus: Clinical, Epidemiological, and Molecular Aspects of a Neglected Orthobunyavirus. \u003cem\u003eThe American journal of tropical medicine and hygiene\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 1019-1030 (2017).\u003c/li\u003e\n\u003cli\u003eFeitoza, L.H.M.\u003cem\u003e, et al.\u003c/em\u003e Influence of meteorological and seasonal parameters on the activity of Culicoides paraensis (Diptera: Ceratopogonidae), an annoying anthropophilic biting midge and putative vector of Oropouche Virus in Rond\u0026ocirc;nia, Brazilian Amazon. \u003cem\u003eActa tropica\u003c/em\u003e \u003cstrong\u003e243\u003c/strong\u003e, 106928 (2023).\u003c/li\u003e\n\u003cli\u003ePAHO. Epidemiological Alert - Oropouche in the Americas Region - 13 December 2024. (2025).\u003c/li\u003e\n\u003cli\u003eMINSAP. Nota informativa del Ministerio de Salud P\u0026uacute;blica 27 de mayo de 2024. (2024).\u003c/li\u003e\n\u003cli\u003eNaveca, F.G.\u003cem\u003e, et al.\u003c/em\u003e Human outbreaks of a novel reassortant Oropouche virus in the Brazilian Amazon region. \u003cem\u003eNature medicine\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eGr\u0026auml;f, T.\u003cem\u003e, et al.\u003c/em\u003e Long-Range Spread and Sustained Transmission of Oropouche Virus Outside the Endemic Brazilian Amazon Region. \u003cem\u003eThe Lancet. Infectious diseases\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eBenitez, A.J.\u003cem\u003e, et al.\u003c/em\u003e Oropouche Fever, Cuba, May 2024. \u003cem\u003eEmerging infectious diseases\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eNaveca, F.G.\u003cem\u003e, et al.\u003c/em\u003e Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses. \u003cem\u003eMemorias do Instituto Oswaldo Cruz\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 510-513 (2017).\u003c/li\u003e\n\u003cli\u003eDeiana, M.\u003cem\u003e, et al.\u003c/em\u003e Full Genome Characterization of the First Oropouche Virus Isolate Imported in Europe from Cuba. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez, Y.M.\u003cem\u003e, et al.\u003c/em\u003e First report of Culicoides paraensis (Goeldi, 1905) (Diptera: Ceratoponidae) in Cuba: A new challenge for public health. \u003cem\u003eParasite epidemiology and control\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, e00423 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7303560/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7303560/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Oropouche virus (OROV) was reported in Cuba in May 2024 and rapidly spread throughout the country. Among 147 RT-PCR-positive cases identified from May to July, we generated 39 whole-genome sequences. Phylogenetic analysis revealed that all sequences formed a monophyletic cluster nested within the novel reassortant OROVBR-2015-2025, which has been circulating extensively in Brazil since the end of 2023. Additional analyses demonstrated that the Cuban sub-clade originated from a single viral introduction from the Brazilian state of Acre, probably in early February 2024, and circulated cryptically until its identification in May. The introduction likely occurred in the Central region of the country, from which the virus spread and established secondary transmission hubs in the Western and Eastern regions. These findings underscore the capacity of OROV to spread well beyond the Amazon region, which was considered its endemic area of circulation.","manuscriptTitle":"Countrywide Spread and Spatiotemporal Diffusion Dynamics of Oropouche virus in Cuba, 2024","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 12:35:27","doi":"10.21203/rs.3.rs-7303560/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nm","sideBox":"Learn more about [Nature Medicine](http://www.nature.com/nm/)","snPcode":"","submissionUrl":"","title":"Nature Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a1f0e119-e700-421d-95ee-a7fe8678a790","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53188308,"name":"Biological sciences/Evolution/Evolutionary genetics"},{"id":53188309,"name":"Health sciences/Medical research/Epidemiology"}],"tags":[],"updatedAt":"2026-04-16T18:30:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 12:35:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7303560","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7303560","identity":"rs-7303560","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}