Marine ecosystems along the coast of Brazil support high biodiversity, including more than 46 recorded cetacean species, several of which are classified as threatened or near threatened. Influenza A viruses (IAV) can infect diverse hosts, including marine mammals. The highly pathogenic avian influenza (HPAI) H5N1 clade has caused widespread outbreaks with high bird and pinniped mortality in South America. Reports of IAV in cetaceans have been rare and limited to a few species, and large-scale cetacean outbreaks have not been described prior to this study.
This surveillance effort screened stranded cetaceans along the Brazilian coastline from November 2023 through August 2025 to detect IAV circulation in marine mammals and to identify possible spillover of HPAI strains into cetacean species, including those already at conservation risk.
Researchers recorded stranded marine mammals along coastlines in three Brazilian states: Rio Grande do Sul (≈485 km), Rio de Janeiro (317 km), and Ceará (573 km). Systematic beach surveys were conducted in Rio Grande do Sul, and teams responded to stranding notifications across coastal regions. For each carcass, standardized metadata were collected, including date, location, species, sex, and decomposition stage.
Swab samples were obtained exclusively from carcasses in decomposition stages 2–4 (bloated stage, active decay, advanced decay). Tissue samples (brain, lungs, trachea, spleen, intestine, liver) were collected and fixed in 10% buffered formalin when carcasses were in earlier decomposition stages suitable for histology. The study received ethical approval from the Animal Ethics Committee of the Universidade do Vale do Rio dos Sinos (protocol no. PPECEUA 02.2025).
Total nucleic acids were extracted using the MagMAX CORE Nucleic Acid Purification Kit with the KingFisher Flex system. Detection of IAV was performed by quantitative reverse transcription PCR (qRT-PCR) targeting the IAV matrix gene. Identification of the H5N1 strain used World Health Organization molecular protocols.
The fluorescence threshold was set at 0.09, and samples with cycle threshold (Ct) values <37 were classified as positive. Assay sensitivity was evaluated using RNA from infected MDCK cells cloned into a plasmid for the IAV matrix assay, and a pEz plasmid with 10-fold serial dilutions to determine the H5N1 qRT-PCR limit of detection. The reported assay limit of detection approached 200–2,000 RNA copies/µL.
A total of 45 stranded cetaceans representing 13 species were analyzed. IAV matrix gene RNA was detected in 18 animals. Species with positive detections included:
Positive detections came from multiple sample types. Blowhole swabs were the most frequent positive sample, followed by cerebral, oral, anal, intestinal, tracheal, and ocular tissue samples.
Overall, detected viral RNA levels were low. Ct values for positive samples ranged from 28 to 37, corresponding approximately to 1.3 × 10^5 down to 8.0 × 10^2 RNA copies/µL. Those copy numbers approach the qRT-PCR assay limit of detection (≈200–2,000 RNA copies/µL).
The HPAI H5N1 strain was detected in 2 samples; both H5N1-positive samples had viral RNA concentrations near the assay limit of detection. Details on which specific tissues yielded H5N1 detections and precise copy numbers were reported in the article’s figures and appendix.
Histopathologic examination was performed on six IAV-positive animals (identified in the appendix as animals 11, 16, 17, 35, 37, and 39). Routine processing of fixed tissues did not reveal lesions associated with influenza infection in those samples. The majority of carcasses were in advanced decomposition stages, which limited the number of specimens suitable for histopathology and likely reduced the chance of observing infection-associated lesions.
IAV detections in cetaceans occurred after major HPAI H5N1 outbreaks in South America that caused high pinniped mortality in 2023, suggesting persistent viral circulation in the marine environment and possible interspecies transmission. Detection of viral RNA in internal tissues supports true infection rather than surface contamination by environmental contact with infected birds. The identification of IAV in species classified as vulnerable (Franciscana dolphin) and endangered (Lahille’s bottlenose dolphin) underscores an additional threat to species already at risk of extinction along the Brazilian coast.
Detection of H5N1 in Franciscana and Clymene dolphins highlights the potential for dissemination of HPAI strains in coastal and offshore waters and indicates a need to monitor impacts on both wildlife conservation and broader marine ecosystem health.
Key limitations affecting interpretation of results include the advanced decomposition stage of most collected carcasses (stages 2–4), which can lead to postmortem RNA degradation and reduce viral detection sensitivity. High Ct values and low copy numbers may partly reflect RNA degradation. The limited number of carcasses available for histopathology reduced the ability to identify lesions associated with influenza infection. Environmental contamination from seabirds and shorebirds cannot be fully excluded, although detection of viral RNA in internal tissues argues for true infection in at least some animals.
Surveillance from November 2023–August 2025 detected influenza A virus RNA in 18 of 45 stranded cetaceans sampled along the Brazilian coast and identified H5N1 in 2 samples with low viral RNA levels. These findings indicate that cetaceans can carry IAV, including HPAI strains, and that marine mammals—some of which are vulnerable or endangered—may be at added risk from viral pathogens. The authors emphasize the importance of continued, systematic surveillance of marine mammals to better understand pathogen dynamics at the wildlife–marine interface and to inform conservation and public health responses.
The study authors acknowledged staff, students, and other contributors who assisted with field and laboratory work. Additional funding sources, collaborator names, and full acknowledgments appear in the article appendix and metadata.