Disclaimer: Initial release articles are not considered final versions. Changes will be reflected in the online version in the month the article is officially released.
Author affiliation: Instituto Conmemorativo Gorgas de Estudios de la Salud, Panama, Panama City, Panama (M. Chen-Germán, C. González, DJ Araúz, M. Vega, O. Chavarria, A. Moreno, E. Santiago, D. Franco, E. Valdespino, J. Gondola, N. Obaldia III, AA Martínez, B. Moreno). University of Panama, Panama City (C. Gonzalez, C. Aguilar). Dr. Hugo Spadafora Polyclinic, Colon City, Panama (M. Pinedo); Panamanian Ministry of Health, Penonome City, Panama (D. Espino, T. Salcedo); Organización Panamericana de la Salud/Organización Mundial de la Salud, Panama City (L. Franco); Gorgas Memorial Health Institute, Panama, Panama (B. Armien)
In recent years, the incidence and geographic range of arboviruses has increased significantly, resulting in multiple epidemics worldwide. Nevertheless, a significant proportion of patients remain undiagnosed, as the primary focus is on clinical suspicion of dengue fever. We report results from an enhanced surveillance initiative in Panama that reanalyzed dengue-negative samples and led to the identification of other viral pathogens, including Oroporchi virus (OROV) and Puntatro virus (PTV). Our results highlight the need to expand diagnostic approaches in arbovirus endemic areas.
OROVs are single-stranded, negative-sense, segmented RNA viruses classified within the family. Peribunyaviridaegenus Orthobunya virusprimarily transmitted by anthropophilic midges. Culicoides paraensis. Olof was first identified in Trinidad and Tobago in 1955 and has spread throughout Central and South America (1). It was first isolated in Panama in 1989, but there is serological evidence of earlier outbreaks in 1968 and 1978 (2).
In 2024, the Pan American Health Organization/World Health Organization reported an increase in OROV cases across the Americas, including areas where there was no previous evidence of an epidemic (3). Health authorities have linked this geographic spread to severe clinical outcomes, including death, vertical transmission, fetal loss, and microcephaly (4). Research in Brazil identified a novel recombination event in the OROV genome (5).
PTV is Fenuiviridae family, phlebovirus Genus. This highly diverse genus includes numerous species distributed throughout the world and transmitted by vectors such as sandflies and ticks (6). Major members include Tuscan virus and Rift Valley fever virus, which cause epidemics and neurological diseases throughout the Mediterranean Basin, Europe, and the Middle East (7). Reports from the Americas revealed that phlebovirus Species from Brazil and Peru (8), Colombia (9) and Panama (10), however, PTV appears to have only been reported in Panama. The human infection, mainly detected in metropolitan areas of Panama, causes a nonspecific febrile illness characterized by fever, headache, weakness, and retroorbital pain (10).
From 2023 to 2024, Gorgas Memorial Institute (Panama City, Panama) analyzed 3,589 serum samples. Following the Pan American Health Organization/World Health Organization warning of rising regional OROV incidence, and due to the lack of confirmed cases in Panama, a subset (24%) of dengue fever virus (DENV)-negative samples from the OROV set was retrospectively tested by reverse transcription-PCR (11). We then integrated this assay into a broader diagnostic algorithm for samples that tested negative for DENV, Zika virus, and chikungunya virus, which included testing for DENV, Zika virus, and chikungunya virus. alphavirus, orthoflavivirusand phlebovirus (Appendix 1).
Through this investigation, we identified OROV positive cases in August 2024. The patient, a 31-year-old male environmental worker stationed in the forest area of Soberanía National Park, Panama, sought treatment for an acute febrile syndrome characterized by severe fronto-occipital headache, chills, arthralgia, and marked asthenia. The man underwent initial clinical evaluation at a primary care facility, where the dengue NS1 antigen test result was negative. The treating clinician reported no neurological symptoms or recurrence. Health authorities conducted contact tracing at the patient’s residence and workplace and collected serum and urine samples from 36 contacts. 12 people with symptoms were infected with OROV, DENV, Zika virus, Chikungunya virus, orthoflavivirus, phlebovirusand alphavirus (Table 1). One of the contacts tested positive. phlebovirus Genus with symptoms such as fever, chills, myalgia, arthralgia, headache, retroorbital pain, and conjunctivitis.
shape
shape. Concatenated maximum likelihood phylogenetic tree of three OROV segments of patients found in the Enhanced Surveillance Detection of OROV and Punta Toro Virus Infections study, Panama, 2023-2024. of…
Both positive samples (OROV and phlebovirus genus) using an in-house metagenomic approach (A. Martinez et al., unpublished data; https://dx.doi.org/10.17504/protocols.io.36wgq6545lk5/v1) (Appendix 1), yielding the complete OROV genome (GenBank accession number PV942050–2) (Table 2). Phylogenetic analyzes were performed using the Nextstrain OROV database (https://github.com/nextstrain/oropouche), we placed the sample at the basal node of the BR-2015–2024 clade (5), which is particularly distinct from the sequences that were widespread in the Brazilian outbreak (shape).
We performed a comparative analysis of OROV using the Panama sequence (hOROV/Panama/A003066/2024), the recent Brazilian sequence (OROV/Saul/17225/2020, LACENAM_ILMD_3228ZCF, ILMD_TF29; GenBank accession number PP154170–2), and the ancestral sequence. Using BeAn19991 stock as a reference. The Panama sequence exhibited multiple amino acid substitutions across structural and nonstructural proteins and shared 96.8% amino acid identity with BeAn19991 in the large (L) segment, 97% to 99% in the medium (M) segment, and 98% to 100% in the small (S) segment. The Panama sequence also shared 99% amino acid identity with the Brazilian sequence in the L segment, 98% to 99% in the M segment, and 100% in the S segment (Appendix 1 Figure 1). We theorized that the difference could affect viral replication or transcription efficiency (KB Gunter et al., unpublished data; https://dx.doi.org/10.1101/2025.08.02.668287).
Upon further inspection, phlebovirus Genus-positive samples as PTV (GenBank accession number PV942053–5) (Appendix 1 Figure 2; Appendix 2). When compared to the prototype strain (GenBank accession number KP272028-30), the amino acid identity was 87% in the L segment, 94% in the M segment, and 95% to 97% in the S segment. Comparison with the 2004 sequence (GenBank accession number KP272031-3) showed 99% amino acid identity with the L segment, 99% with the M segment, and 98% to 99% with the S segment (Appendix 1 Figures 3, 4).
Before the OROV outbreak that began in Panama in January 2025, the last recorded case occurred in 1989 in Bejuco, a coastal region approximately 55 miles west of Panama City, which at the time was primarily surrounded by mature hardwood forest (2). The OROV cases we identified may have been infected in a similar ecological setting at the border between the metropolitan area and Soberanía National Park. The geographical proximity (22–25 miles) between Bejuco and Soberanía National Park suggests common ecological features and supports the hypothesis that the cryptic OROV circulation was continuing before the 2025 cases were reported in Darien Department.
Although decades of missing genomic data have limited reconstruction of the complete evolutionary history of OROV in Panama, this case provides valuable evidence suggesting a persistent, undetected sylvatic circulation. The Panama OROV sequence shows significant amino acid differences from the ancestral BeAn19991 strain, especially in the L and M segments, and shows fewer differences compared to the strains currently circulating in Brazil and South America reported since 2022 (5). These changes are similar to mutations reported in recent studies (GC Scachetti et al., unpublished data; https://dx.doi.org/10.1101/2024.07.27.24310296). Differences between the Panama strain and the newly described OROV reassortants suggest an ongoing adaptive process potentially driven by local selective pressures or long-term latent cycles in Panama. Sporadic retrospective OROV detections from dengue surveillance samples in Brazil reflect this situation (12).
PTV (10) is an arbovirus that appears to be underreported in Panama, complicating the clinical suspicion required for accurate diagnosis. Our sequenced samples revealed considerable genetic diversity compared to the reference strain, but less divergence compared to more recent sequences, suggesting both divergence from the original reference strain and ongoing virus evolution.
Both of the patients we describe worked in areas of mature hardwood forest, suggesting that this is the site of infection and indicating that different arboviruses may potentially be circulating at the same time. The enhanced arbovirus surveillance described in our study included testing across multiple virus genera, expanded analysis of dengue-negative samples, and enabled detection of other arboviruses. Of note, the OROV and PTV strains in our study showed greater genetic similarity among recent strains, indicating ongoing evolution, whereas divergence from older strains reflects the accumulation of mutations over time. Taken together, these findings highlight the complexity and dynamic nature of arbovirus evolution and highlight the critical need to strengthen and expand arbovirus surveillance frameworks beyond routine detection of dengue to encompass the full range of circulating arboviruses and their potential impact on public health.
Ms. Chen German is a medical technician at Gorgas Memorial Research Institute, specializing in arboviruses.