Fig 1.
Study area, sampling points in southeastern Brazil.
The map highlights the six sampling sites in the northeast of São Paulo State. Areas with native vegetation of Cerrado patches and Atlantic Forest enclave are represented in green. In yellow are agricultural areas, and in grey is urbanization. The map was generated by QGIS 3.26.0 (Zürich-CH). The base layer used in this map are from Instituto Brasileiro de Geografia e Estatística (IBGE) https://www.ibge.gov.br/geociencias/downloads-geociencias.html.
Table 1.
Sample number of Orthoflavivirus (RT-PCR) and Alphavirus (RT-PCR and serology) in collected NHP.
Table 2.
Distribution of tested animals according to sex, age classes, and seasonality.
Table 3.
NHP species tested for Orthoflavivirus and Alphavirus, and distribution according to sex and age classes.
Table 4.
Orthoflaviviruses identified by amplicon according to the NHP species, including data on viral isolation.
Fig 2.
Saliva from non-human primates contains infectious Orthoflavivirus louisense (SLEV) detectable by indirect immunofluorescence assay (IFA) in VERO and C6/36 cells.
Viral protein labeling (green) was detected using polyclonal antibodies against SLEV, and nuclei were counterstained with DAPI (blue). Panels A and C show uninfected VERO cell controls (CN). Panels B and D display VERO cells infected with SLEV-positive saliva samples from Saguinus midas (B) and Alouatta caraya (D), respectively. Panel E shows uninfected C6/36 cell controls (CN), and panel F shows C6/36 cells infected with SLEV-positive saliva from Sapajus apella. White arrows indicate areas of viral protein labeling (green). Cells were fixed and processed for IFA five to six days post-infection. Scale bars: 50 µm (A-D, F) and 100 µm (E).
Fig 3.
Phylogenetic analysis reveals that the isolated Orthoflavivirus denguei is closely related to Orthoflavivirus denguei 3 (DENV-3) genotype III, major lineage C.
Maximum-likelihood phylogenetic tree of DENV-3 based on the NS5 gene segment. SH-aLRT support values are indicated at the branch nodes. The tree was inferred using the HKY85 + F nucleotide substitution model. Sequences in the branch highlighted in dark blue represent Brazilian DENV-3 isolates. In contrast, the sequence obtained in this study (GenBank accession no. OQ032518.1) corresponds to DENV-3 genotype III, major lineage C, and is highlighted in blue. The sequence OK469356.1 was used as an outgroup reference.
Fig 4.
Eco-epidemiological species-related differences of Orthoflavivirus infection prevalence among non-human primates in southeastern Brazil.
Prevalence ratios (95% confidence intervals) for Orthoflavivirus infection were estimated based on morphometric, behavioral, sex, age, and species characteristics of non-human primates (NHPs). Variables included allometric cluster (Cluster 1 = animals with higher allometric measurements; Cluster 2 = animals with lower measurements), presence of wounds (Yes/No), sex, age category, and species (Sapajus apella, Alouatta caraya, and Callithrix penicillata). A significantly higher infection prevalence was observed in S. apella and A. caraya compared with C. penicillata (p < 0.01). CI = confidence interval; vs = versus.
Fig 5.
Spatial distribution and density of Orthoflavivirus-infected non-human primates (NHPs) in Ribeirão Preto County, São Paulo State, Brazil.
Kernel density estimation (KDE) risk map showing the spatial distribution and density of Orthoflavivirus-infected NHPs (red circles) in Ribeirão Preto County. Mapping and spatial analyses were performed in QGIS version 3.26.0 using distance matrices between confirmed arbovirus cases. The quartic kernel function was applied, providing higher weighting to nearby points and lower weighting to distant ones, with bandwidth values of 2,562.09 for the complete dataset and 3,106.44 when considering only the genus of interest. A continuous KDE classification was employed for improved visual representation. Built-up areas, agricultural zones, and forest fragments are indicated in grey, beige, and green, respectively. Base layers were obtained from the Instituto Brasileiro de Geografia e Estatística (IBGE; https://www.ibge.gov.br/geociencias/downloads-geociencias.html).