Silent Footprints of Ebolavirus in the Forest: Serological Clues from Wild Non-Human Primates in the Democratic Republic of Congo

Abstract Filoviruses, particularly Ebola virus (EBOV), remain a major public health concern in Central Africa. However, their circulation in wildlife during inter-epidemic periods remains poorly documented. Non-human primates (NHPs) may serve as ecological sentinels of viral dynamics at human-forest interfaces, yet surveillance is constrained by the limitations of invasive sampling. We conducted a non-invasive investigation of EBOV exposure in free-ranging NHPs from the Mabali Forest Reserve (Equateur Province, Democratic Republic of Congo) for Ebola virus disease. A total of 630 fecal samples were collected and screened for active infection by PCR targeting the EBOV nucleoprotein gene; all samples tested negative. Molecular identification of host species was achieved in 569 samples (90.3%). Fecal serology using an automated capillary western blot platform (JESS), targeting EBOV nucleoprotein, glycoprotein and viral protein 40 antigens, identified four seropositive individuals (0.70%), including two Cercopithecus ascanius and two C. wolfi . The detection of discrete immunoreactive bands consistent with EBOV-specific antibodies suggests prior exposure despite the absence of active outbreaks. These findings provide the first serological evidence compatible with EBOV exposure in these two Cercopithecus species and support the hypothesis of low-level or cryptic viral circulation in forest ecosystems. The study highlights the feasibility and value of fecal serology as a non-invasive One Health surveillance tool for monitoring zoonotic pathogens at wildlife–human interfaces.