# Fire-footed rope squirrels (Funisciurus pyrropus) are a reservoir host of monkeypox virus (Orthopoxvirus monkeypox)

Fabian H Leendertz, Carme Riutord-Fe, Jasmin Schlotterbeck, Lorenzo

Lagostina, Leonce Kouadio, Harriet Herridge, Moritz Jochum, Nea Noma, Ane López-Morales, Donata Hoffmann, et al.

► To cite this version:

Fabian H Leendertz, Carme Riutord-Fe, Jasmin Schlotterbeck, Lorenzo Lagostina, Leonce Kouadio, et al.. Fire-footed rope squirrels (Funisciurus pyrropus) are a reservoir host of monkeypox virus (Orthopoxvirus monkeypox). 2025. hal-05043628

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# Fire-footed rope squirrels (*Funisciurus pyrropus*) are a reservoir host of monkeypox virus (*Orthopoxvirus monkeypox*)

## Fabian Leendertz

fabian.leendertz@helmholtz-hioh.de

Helmholtz Institute for One Health, Helmholtz Center for Infection Research https://orcid.org/0000-0002-2169-7375

## Carme Riutord-Fe

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

## Jasmin Schlotterbeck

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

## Lorenzo Lagostina

Helmholtz Institute for One Health, Helmholtz Center for Infection Research https://orcid.org/0000-0002-6661-705X

## Leonce Kouadio

Tai Chimpanzee Project, Centre Suisse de Recherches Scientifiques en Côte d'Ivoire
https://orcid.org/0000-0003-1819-3333

## Harriet Herridge

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

## Moritz Jochum

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

## Nea Noma

Senckenberg Museum of Natural History, Senckenberg Society for Nature Research

## Ane López-Morales

Tai Chimpanzee Project, Centre Suisse de Recherches Scientifiques en Côte d'Ivoire

## Donata Hoffmann

Friedrich Loeffler Institute https://orcid.org/0000-0003-4552-031X

## Sten Calvelage

Friedrich Loeffler Institute

## Hjalmar Kühl

Senckenberg Museum for Natural History Görlitz, Senckenberg-Member of the Leibniz Association

## Alexander Mielke

Tai Chimpanzee Project, Centre Suisse de Recherches Scientifiques en Côte d'Ivoire
**Catherine Crockford**

French National Centre for Scientific Research

**Roman Wittig**

Tai Chimpanzee Project, Centre Suisse de Recherches Scientifiques en Côte d'Ivoire

**Martin Beer**

Friedrich-Loeffler-Institute https://orcid.org/0000-0002-0598-5254

**Gonedelé Sery Ernest**

UFR Biosciences, Université Félix Houphouët Boigny

**Jan Gogarten**

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

**Sébastien Calvignac-Spencer**

HIOH https://orcid.org/0000-0003-4834-0509

**Ariane Dux**

Robert Koch Institute

**Livia Patrono**

Helmholtz Institute for One Health, Helmholtz Center for Infection Research

**Biological Sciences - Article**

**Keywords:**

**Posted Date:** April 8th, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6322223/v1

**License:** © This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is **NO** Competing Interest.
Abstract

Mpox, caused by the monkeypox virus (MPXV; *Orthopoxvirus monkeypox*), is on the rise in West and Central Africa. Most outbreaks are short-lived, but MPXV has recently caused larger epidemics driven by sustained human-to-human transmission. It is widely accepted that mpox outbreaks originate in zoonotic events. African rodents, especially squirrels, are suspected to be involved in MPXV emergence, but no formal link to human or nonhuman primate outbreaks has been established. Here, we describe an outbreak of MPXV in a group of wild sooty mangabeys (*Cercacebus atys*) in Taï National Park (Côte d'Ivoire). The outbreak affected one third of the group between January and April 2023, killing four infants. To track its origin, we analysed rodents and wildlife carcasses from the region. We identified a MPXV-infected fire-footed rope squirrel (*Funisciurus pyrropus*), found dead 3 km from the mangabey territory 12 weeks before the outbreak. MPXV genomes from the squirrel and the mangabey were identical. To establish a potential link between these species, we investigated the diet of these mangabeys. We found one video record of consumption of the same squirrel species in 2014. In addition, we performed metabarcoding analyses of faecal samples collected from mangabeys in the four months prior to the outbreak, which identified two faecal samples containing the DNA of the fire-footed rope squirrel. One of these samples was also the first positive for MPXV in the mangabey group. This represents an exceptionally rare case of direct detection of an interspecies transmission event, made possible only by long-term health monitoring. Our findings strongly suggest rope squirrels were the source of the MPXV outbreak in mangabeys. Since squirrels and nonhuman primates are hunted, traded, and consumed by humans in West and Central Africa, exposure to these animals is likely responsible for at least a fraction of human mpox outbreaks.

Main

The recent emergence of MPXV lineages characterized by human-to-human transmission through sexual
networks has led the WHO to declare public health emergencies of international concern in 2022¹ and
2024². Efforts were immediately scaled up in endemic African countries to reinforce mpox surveillance
systems. Concurrently, sustained human-to-human transmission was shown to leave a distinct signature
in MPXV genomes under the form of APOBEC3-induced mutations, providing a potent tool to determine
how much of MPXV evolution happened in humans³. Building on these two pillars, recent genomic
surveillance data from the Democratic Republic of Congo (DRC) and the Republic of Congo clearly
showed that MPXV diversity mostly reflects numerous, independent zoonotic spillovers⁴,⁵. Importantly,
epidemiological data from the DRC suggest that these spillovers have increased in frequency from 2010
to 2024, a period during which the national surveillance system has been relatively stable⁶. Although
African rodents have long been considered a possible reservoir for MPXV⁷,⁸, formal proof to support this
assumption is lacking. Ascertaining the animal sources of zoonotic agents is indeed a notoriously
difficult task⁹. In the case of MPXV, achieving this task is critical, since it would lead to better
management of the risk of spillover to humans, and thereby provide a lever to prevent subsequent
outbreaks fueled by human-to-human transmission.
Captive nonhuman primates (NHP) are famously associated with the discovery of MPXV¹⁰, which resulted in the misleading naming of the virus. More recently, long-term health monitoring at the Taï Chimpanzee Project¹¹ (TCP) in Taï National Park (TNP), Côte d'Ivoire, revealed that MPXV also affects wild NHP, opening a window into the ecology of this virus. We detected MPXV in a dead sooty mangabey (hereafter mangabey; *Cercacebus atys*) in 2012¹², and later identified three independent MPXV outbreaks in 2017 and 2018 that hit distinct groups of western chimpanzees (*Pan troglodytes verus*) living in the same forest¹³. While these investigations provided insights into different clinical presentations and described viral genomic diversity in this area, the source of these outbreaks remains unknown. In late January 2023, we started observing clinical signs compatible with MPXV infection in several infants from the habituated mangabey group of TCP, which consisted of 80 individuals at that time (Fig. 1). Nearly 30% of the group developed skin lesions over the following 12 weeks. Making use of TCP's longitudinal non-invasive sample collection started in the early 2010s¹⁴, along with the systematic carcass monitoring¹⁵,¹⁶, and rodent sampling efforts in and around TNP, we conducted an outbreak investigation that included an extensive search for a potential source of infection.

On January 27th 2023, an infant mangabey developed red macular lesions on the forehead, back of the head, chest and legs (Fig. 2A), accompanied by the onset of lethargy and anorexia. Lesions quickly spread to the entire body and the individual died within 48 hours, on January 29th. By early March, five other infants developed similar lesions alongside lethargy, anorexia, and lymphadenopathy. Macular skin lesions progressed to papulo-pustular stages, and three of these infants died. A milder form of the disease, consisting of either a diffuse rash with only ~5-20 skin lesions (Fig. 2B), or fewer isolated lesions appearing in a single part of the body (e.g. face, limbs or tail, Fig. 2C), affected 20 other mangabeys of all age groups (Extended Data Fig. 1). In all affected animals, papulo-pustular lesions evolved to crusts and ultimately scabs (Extended Data Fig. 2). Overall, the disease swept through the group until the end of April 2023, resulting in 26 out of 80 (32.5%) mangabeys developing at least one visible skin lesion, and four deaths. Trained veterinarians wearing a complete set of personal protective equipment and following strict biosafety protocols performed on-site necropsies on three out of the four infants. The body of the fourth infant was never found.

To confirm infection with MPXV, we first tested necropsy samples from the three infants and identified viral DNA in all major organs (Supplementary Table 1). We then performed a group-wide outbreak investigation by analyzing 170 faecal samples collected from the mangabeys during the outbreak window, defined as the period in which clinical signs were visible in the group (Extended Data Fig. 3 and Supplementary Table 2). We detected MPXV DNA in 36 faecal samples collected from 19 individuals (7 symptomatic and 12 asymptomatic, Fig. 3). Of these 19, 14 were mothers of symptomatic babies, and only six of them developed lesions (Supplementary Table 3). We did not detect MPXV in 89 faecal samples collected after clinical signs resolved. These findings show that MPXV caused disease in a large proportion of this group and may have infected an even larger pool of individuals subclinically, consistent with earlier observations in chimpanzees from TNP¹³.
To reconstruct viral genomes and determine their relationships with MPXVs that previously emerged in this area, we applied hybridization capture coupled to high-throughput sequencing to both necropsy and faecal samples. This allowed us to assemble two complete genomes, from a necropsy sample collected on January 31st 2023 (371x average depth of coverage) and a fecal sample collected on February 12th 2023 (22x). The two genomes were identical, and maximum-likelihood phylogenetic analyses placed them within the genetic diversity of clade IIa viruses, as close relatives to the other MPXVs from TNP (Fig. 4). This suggests that this outbreak was the result of a transmission event involving the same local reservoir(s).

Multiple species of squirrels (*Funisciurus* sp., *Heliosciurus* sp.) and other rodents, such as Gambian pouched rats (*Cricetomys gambianus*) and African dormice (*Graphiurus* sp.) are suspected to be involved in the sylvatic maintenance of MPXV (reviewed in ref⁸). To investigate whether squirrels or other small terrestrial mammals could be a source of MPXV infection for the TNP non-human primates, we tested rodents and shrews trapped (n=694) or found dead (n=10) inside and around TNP between 2019 and 2024 (Supplementary Tables 4 and 5). We identified one MPXV-positive fire-footed rope squirrel found dead on November 3rd 2022 – 12 weeks before the onset of the outbreak in the mangabeys, and approximately 3 km south of their territory (Extended Data Fig. 4). Due to the opportunistic nature of our sampling, we could not determine whether the squirrel died of MPXV infection or due to other causes (e.g. predation; necropsy report available in the Supplementary Information). All organs from the squirrel (n=15), as well as oral and nasal swabs, contained high viral loads, and we were able to isolate viable MPXV from the skin, lung, spleen and liver of the animal (Supplementary Table 5). We also sequenced the complete genome of this virus (114x from samples, 20x from the lung isolate) and found it to be identical to the MPXV genomes derived from the mangabey samples. Since the isolation of MPXV from a Thomas's rope squirrel (*Funisciurus anerythrus*) in 1978¹⁷, only relatively weak evidence has accumulated supporting African squirrels as natural hosts of MPXV⁷,¹⁸,¹⁹. Our findings considerably reinforce the hypothesis that fire-footed rope squirrels are natural hosts of MPXV. The exact same genomic MPXV variant was circulating in the local squirrel population in the weeks preceding the outbreak in the mangabeys, a first hint that squirrels may have served as the reservoir host.

We then aimed at refining a plausible scenario of MPXV emergence. For this, we first tested 114 faecal samples collected from the mangabey group in the 16 weeks before the outbreak. We identified 10/114 MPXV positive faecal samples (detection rate 8.8%; 95% CI 4.8-15.9). MPXV DNA was present in faecal samples of seven mangabeys that were asymptomatic at the time of detection, including the mother of the first infant to show clinical signs (Fig. 3). Importantly, the three earliest positive samples were consecutively obtained from the same individual, on December 6th, 9th and 18th, 2022. Collectively, these results suggest that MPXV entered the group via this plausible index case and then circulated undetected in the group for nearly two months.

Mangabeys are known to hunt small mammals, including in TNP. Reviewing available long-term behavioural data, we found a video recording from 2025 showing a mangabey catching a squirrel
(Supplementary Video 1), and an older video from 2014 showing a mangabey feeding on a clearly identifiable fire-footed rope squirrel (Fig. 5 and Supplementary Video 2). To explore whether a fire-footed rope squirrel hunt may have been the source of this outbreak, we analyzed the mangabey diet before the outbreak by searching for prey DNA in the 78 earliest faecal samples of our collection (Supplementary Table 2). Using mammal-generic metabarcoding, we identified DNA sequences perfectly matching the mitogenome of the TNP fire-footed rope squirrel in two faecal samples (Supplementary Information). This demonstrates that these squirrels are not only part of the mangabey diet, but that group members fed on this species on at least two distinct occasions in the weeks before the outbreak. Even more strikingly, we found that one of the two samples containing squirrel DNA was also the first MPXV-positive faecal sample of the suspected index case (Fig. 3). The co-detection of squirrel and MPXV DNA in this faecal sample strongly hints at an exceptional case of real-time detection of a cross-species transmission event, which subsequently led to the group-wide MPXV outbreak. Based on all the accumulated evidence, we conclude that fire-footed rope squirrels serve as a reservoir of MPXV for wild nonhuman primates in TNP.

Bushmeat remains an important source of protein in sub-Saharan Africa, including Côte d'Ivoire. In many regions, the recent decline of large-bodied mammals due to habitat destruction and hunting has induced a shift in consumption towards smaller animals, especially rodents<sup>20, 21-23</sup>. A study conducted in the villages bordering TNP has shown that while primates remain the most hunted taxa, rodents are also commonly traded and consumed<sup>24</sup>. Although larger rodents such as the giant pouched rat (*Cricetomys* sp.) and the marsh cane rat (*Thryonomys swinderianus*) are more frequently seen on markets, multiple species of squirrels are also sold and consumed, in both rural and urban areas<sup>25</sup> (unpublished data from N.Y.N., H.K. and S.G.-B.). It is also worth noting that, contrary to nonhuman primates who mainly rely on intact forest ecosystems, squirrels can thrive in fragmented habitats and plantations close to villages<sup>26</sup>. Upon re-analyzing a mammal-generic metabarcoding dataset derived from carrion flies collected along a gradient from pristine forest to the surrounding villages at the same site<sup>27</sup>, we only detected fire-footed rope squirrels in secondary forests and plantations, suggesting a higher presence of this species in these habitats (Supplementary Table 6). In such areas squirrels are commonly trapped by the local population, including children, and directly consumed (personal communication, N.Y.N. and S.G.-B.). Both subsistence hunting and bushmeat hunting, trade and consumption may result in MPXV transmission to humans.

Research on the ecology, habitat use and population dynamics of fire-footed rope squirrels, as well as the dynamics of MPXV infections in these populations and their interactions with humans, will be key to assessing spillover risks from this species. Efforts aimed at identifying other small mammal species that may serve as natural hosts and reservoirs should also be continued, since the involvement of several host species is possible<sup>28</sup>. At the same time, MPXV genomic surveillance in humans in endemic areas remains the most abundant source of information on this virus' diversity in its reservoir(s). For example, we observed that genomes published from human mpox cases caused by clade IIa MPXVs in Côte d'Ivoire in 2024, including two from a town ~80 km north of our study site, were not closely related
to the ones circulating in TNP wildlife (Fig. 4). This confirms a large strain diversity, even at small spatial scales, and perhaps suggests geographic structure and evolution in distinct hosts, though further studies are needed to test this hypothesis conclusively. Importantly, a better understanding of MPXV ecology will inform local authorities in charge of public health, animal health, as well as protected areas and natural resources, and help them develop programs to assess and mitigate spillover risk. This might include campaigns to raise awareness about the general risks linked to bushmeat and initiatives to co-design measures specifically aimed at reducing contact with squirrels through subsistence hunting. The example of TNP shows (1) the direct link between a rodent reservoir and a spillover host and (2) that both may be sources of human infections, suggesting a focus on squirrel consumption alone would be misguided.

This study underscores the high value of long-term wildlife health monitoring<sup>29,30</sup>. While challenging to fund and implement, such projects have a unique ability to unveil interspecies transmission and zoonotic emergence in complex environments, and ultimately offer crucial insights for global public health.

## Declarations

Research was conducted under research permit numbers: 006/MESRS/DGRI (TCP 2022-2025), 461/MINEDD/OIPR/DT, 020/MESRS/DGRI and 007/ MESRS/DGRI (small terrestrial mammal trapping

## Acknowledgements

We thank the Ministère de l'Enseignement Supérieur et de la Recherche Scientifique, the Ministère de Eaux et Fôrets in Ivory Coast and the Office Ivoirien des Parcs et Réserves for permitting the study. We are grateful to the Centre Suisse de Recherches Scientifiques en Côte d'Ivoire and the staff members of the Taï Chimpanzee Project for their support. We thank all primatologists, research assistants, field assistants, and veterinarians who collected samples for the Taï Chimpanzee project over the years. We acknowledge the contribution of Arsène Mossoun, Valere Kouakou Kouamé, Emmanuel Kemin Diané, Privat Gnabro Hugues, Assemian Krou Hermann, Koukou Youl Sie, Mathurin Yao Koffi, Michèle Djê bi Tah, Fabrice Fernand Logoué, Kamilla Anna Pléh, Jonas Steiner, and Olivia Dimov to the small mammal trapping in TNP. We are grateful to Manon Digoin Danzin for providing the video of the managbey catching a squirrel in TNP. This work was supported by the German Research Council Project LE1813/11-1 and LE1813/14-1 (Great Ape Health in Tropical Africa), the ARCUS Foundation grant G-PGM-2107-3516, the BIODIV-AFREID Project LE1813/17-1, the Heinrich Böll Stiftung PhD stipend to J.S., the Evolution of Brain Connectivity Project of the Max Planck Society (M.IF.EVAN8103). Research was conducted under research permit numbers: 006/MESRS/DGRI (TCP 2022-2025), 461/MINEDD/OIPR/DT, 020/MESRS/DGRI and 007/ MESRS/DGRI (small terrestrial mammal trapping).

## Contributions

Data and samples from the outbreak were collected by C.R.-F., A.L.-M and the TCP field assistants and research assistants. Data and samples from the rodents in TNP were collected by L.L., L.K., J.S., M.J.S.J.
and H.R.H. The field investigations as well as diagnostic and research activities were coordinated by S.C.-S., A.D., L.V.P. and F.H.L. Molecular laboratory analyses were performed by C.R.-F, J.S., H.R.H., L.L., A.D. and L.V.P. Diet analyses were performed by C.R.-F and J.F.G. Virus isolation experiments and sequencing of the isolates were conducted by J.S., D.H., S.C., and coordinated by M.B. N.Y.N., H.K. and S.G.B. provided information on bushmeat consumption in Côte d'Ivoire. R.M.W., C.C., A.D. and F.H.L. coordinated the mangabey field work and provided the behavioural data. J.F.G., A.M. and R.M.W provided the video. The data were analysed by C.R.-F., J.S., L.V.P. and S.C.-S. and the manuscript was drafted by C.R.-F., J.S., S.C.-S., L.V.P and F.H.L. The manuscript was revised and approved by all authors.

**Competing interests**

The authors declare no competing interests.

**Additional information**

Supplementary Information is available for this paper.

**Corresponding author**

Correspondence and requests for materials should be addressed to Livia V. Patrono and Fabian H. Leendertz.

**Data availablity**

All sequences generated in this study are currently being submitted to the European Nucleotide Archive.
Accession numbers will be provided before publication. In the meantime, we provide provisional access
to all materials on Zenodo (https://zenodo.org/records/15111982).

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## Methods

**Health monitoring and sampling.** Taï National Park (TNP), Ivory Coast, is the largest remaining primary rainforest in Western Africa. Its wild populations of non-human primates (NHP) have been studied by the Taï Chimpanzee Project (TCP) since 1979³¹. TCP established a veterinary programme from 2001 onwards¹³. This veterinary programme conducts wildlife mortality surveillance and health monitoring of the four neighbouring groups of chimpanzees and one group of sooty mangabeys that are habituated to human observers. The mangabey group (named the Audrenisrou group) was habituated in November 2012³², and at the time of the outbreak consisted of about 80 individuals. The habituated groups are followed daily by trained field assistants and research staff. Names are given to each habituated
individual. Newborns are given a temporary name indicating that they are the infant (BB) of a certain
individual (e.g. BB-Atacama indicates the newborn of Atacama). Behavioural data, as well as faecal and
urine samples, are routinely collected from all the adults of the group. Faecal samples are collected with
a plastic spatula right after defecation occurs and stored in 2 mL cryotubes. Urine is collected with fine
Pasteur pipettes from underlying vegetation once the animals urinate from a higher position, and is
stored in 2 mL cryotubes. These samples are preserved in liquid nitrogen in the field, transported to
Germany in dry ice and then stored at -80°C until further analysis. When clinical signs are observed in the
groups, observations and sampling are intensified. During this MPXV outbreak faecal samples were
collected in both dry 2 mL cryotubes and in cryotubes containing nucleic acid preserving (NAP) buffer
from most individuals of the group belonging to all age categories (infants, juveniles, subadults and
adults), and from both symptomatic and asymptomatic individuals. Faecal samples are difficult to
collect from infants, therefore the number of these samples is lower than other age classes. It is also
important to note that in 2022 a significant number of male juveniles immigrated to the Audrenisrou
group, and many births occurred, leading to an increase in the total population to 80 individuals. To
obtain an overview of viral DNA shedding in the mangabey group, we tested faecal samples from three
key time periods: from four months prior to the first observations of clinical signs (October 1st 2022 -
January 26th 2023), during the outbreak (January 27th 2023 - April 26th 2023), and up to four months
after the last symptoms were observed (April 27th 2023 - August 24th 2023). A total of 444 faecal
samples were tested for MPXV, including those from just before (n = 114), during (n = 170) and after (n =
89) the mpox outbreak in the mangabey group. Details are provided in Supplementary Table 2. The
veterinary team of TCP also performs necropsies on all animals found dead within the research area.
Necropsies are done by trained veterinarians wearing full personal protective equipment. All used
materials are incinerated or disinfected with 1% sodium hypochlorite solution and the carcasses are
buried, according to the World Health Organization guidelines. Samples are collected from all inner
organs when carcass decomposition is not too advanced, and stored in 2 mL cryotubes, both empty and
filled with NAP buffer. The cryotubes are then preserved in liquid nitrogen in the field, transported to
Germany in dry ice, and stored at -80°C until further analysis. In this study we included 88 necropsy
samples from 23 carcasses representing 11 species (and 4 species for which taxonomic assignment
was not possible) collected between 2019 and 2024. Further details are provided in Supplementary
Tables 1 and 5.

**Small terrestrial mammal trapping inside and around TNP.** Rodents and shrews were trapped using Sherman, Havahart-style or 0.5 m cage traps and were anesthetized using a combination of Ketamine (mouse dose 50 mg/Kg, rats dose 35 mg/Kg, Medistar®) and Xylazine (mouse dose 5 mg/Kg, rat dose 3,5 mg/Kg, WDT®) intramuscularly. Once anesthetized, the animals were measured, weighted and sampled. After sampling, the animals were marked and placed in an individual box until full recovery was observed. Anesthetized animals were monitored closely and in some cases Antisedan (5 mg/Kg, Vetoquinol®) was administered intramuscularly to facilitate recovery. Saline solution was sometimes applied as a subcutaneous infusion to prevent dehydration and applied on the eyes to prevent from drying. After recovery, the animals were released where they were caught. From July to November 2021
and March to April 2023, 173 rodents were trapped within the territory of the mangabey group. From these two field missions, we collected 167 oral swabs, 167 rectal swabs, 23 nasal swabs, 133 faecal samples and 39 samples from skin lesions. Eight individuals died in the trap or succumbed to anaesthetisation, and one was euthanized because of signs of extreme weakness. In these cases, necropsies were performed and samples were collected from all major organs. All samples were stored in 2 mL cryotubes dry or with NAP buffer, frozen in liquid nitrogen in the field, transported in dry ice to Germany and then kept at -80°C. From these trapping missions, a total of 553 samples, including different tissues and swabs were tested for orthopoxviruses (OPV). We also made use of samples originating from a broader initiative aimed at characterizing the biodiversity of small mammals and related pathogens along a gradient spanning from three villages bordering TNP on the West to the pristine forest in the immediate vicinity of the sooty mangabey territory. We set traps along three parallel transects of ~9 km covering distinct environments: (i) anthropic/domestic (inside houses), (ii) at the village periphery, (iii) at the edge between cultivated fields and the national park, and (iv) in the pristine forest of the national park. Sampling was performed from July to September 2021 and April to May 2022. In total, 521 rodents and shrews were trapped and sampled (as mentioned above), of which 79 were euthanised and underwent a full necropsy. Oral and rectal swabs were stored in 2 mL tubes with NAP buffer at room temperature until transport to Abidjan where they were stored at -20°C. Necropsy samples were stored in 2 mL cryotubes and immediately frozen in liquid nitrogen. Samples were transported to Germany on dry ice and then stored at -80°C (necropsies) or -20°C (swabs in NAP buffer). From this sample set, we tested 521 oral swabs, 257 rectal swabs, and different organs from the 79 necropsies. *In toto*, 1035 samples from different tissues and swabs were tested for OPV. Details regarding the sampled animals are provided in Supplementary Table 4.

**DNA extraction and orthopoxvirus DNA detection.** Nucleic acids were extracted from 40 mg of faecal matter using the GeneMATRIX Stool DNA Purification Kit (Roboklon, Poland). For the necropsy samples, 20 mg of tissue were used for DNA extraction with the DNeasy Blood and Tissue kit (Qiagen, Germany) or QIAamp Viral RNA Mini Kit (Qiagen). Nucleic acids from virus isolates were extracted using the NucleoMag VET Kit (Macherey-Nagel, Germany) and with the RNAdvance Tissue Kit (Beckman Coulter, USA). DNA extracted from faecal and necropsy samples (excluding rodents) was tested for MPXV in duplicates with a TaqMan Real-time quantitative PCR (qPCR) targeting G2R loci of monkeypoxviruses³³. Rodent DNA extracts (including the DNA extracts from trapped rodents and necropsies) were tested in duplicate for orthopoxviruses using a TaqMan Real-time PCR targeting the P4A gene³⁴ and the positive extracts were tested with the MPXV qPCR mentioned above. A confirmatory PCR targeting a 270-bp fragment of the hemagglutinin (HA) gene of OPVs³⁵ was performed for all the extracts that had weakly positive results in the MPXV or OPV qPCRs.

**Virus isolation.** Virus isolation was attempted from 13 faecal samples (12 from the mangabeys, 1 from the fire-footed rope squirrel), 13 tissue samples, and maggots from two necropsies. Skin, lung, and spleen were tested for each mangabey necropsy, as well as a maggot from one individual. The squirrel samples tested encompassed skin, lung, spleen, liver, faeces, and maggots. The samples were added to
cell culture medium with 10% foetal bovine serum supplemented with penicillin/streptomycin (Gibco, USA) and gentamicin/amphotericin (Gibco), bead homogenised on a bead ruptor, and incubated over night at 8°C. Sample homogenate was filtered through a 0.8µm pore membrane to remove larger particles and potential contaminating bacteria. The filtrate was added to confluent layers of MA-104 cells, and cultivated with the aforementioned antibiotic-supplemented medium in 12.5cm² rectangular canted neck cell culture flasks. Cell cultures were passaged after three days. If a cytopathic effect was visible, cells were passaged further to increase the viral titre for shotgun sequencing.

**Hybridization capture and high-throughput sequencing.** Illumina-compatible dual index libraries were generated from up to 1000 ng of DNA extracts from necropsies and four mangabey faecal samples (details provided in Supplementary Tables 1, 2 and 5). Faecal samples were selected based on viral copy number. DNA was fragmented in 50 µl of low EDTA-TE buffer using a Covaris ME220 Focused-ultrasonicator (Covaris, USA) set for a target fragment-size of 350bp (settings: treatment duration 45 s, peak power 50, duty factor 20%, 1000 cycles per burst, average power 10, temperature 20 °C). Libraries were built from the fragmented DNA using the NEBNext Ultra II DNA kit following the manufacturer's recommendations. After the adapter ligation, a 300 – 400 bp size selection using MagSi magnetic beads (Carl Roth, Germany) was performed where the input was higher than 50 ng of total DNA. Quantification of the final libraries was performed using the Kapa HiFi Library Quantification Kit (Roche, Switzerland) or the NEBNext Library Quant Kit for Illumina (New England Biolabs, USA). Libraries were stored at -20°C until further use. All libraries underwent MPXV target enrichment through in-solution hybridization capture with a previously described orthopoxvirus bait set¹⁰. We used myBaits® RNA baits following the myBaits® Sequence Enrichment for Targeted Sequencing protocol (Version 5.0; Daicel Arbor Biosciences) and applied 2 successive rounds of 24h hybridization capture. Additionally, one round of 24h 65°C hybridization capture targeting the mitochondrial genome of rodents was performed on a library of the squirrel spleen. To design these custom baits, all complete mitochondrial genomes of rodents available on GenBank were accessed and redundancies were reduced by clustering genomes using CD-HIT30 at a minimum of 88% sequence identity. Final bait design was based on the resulting 239 accession numbers. For capture, only a quarter of the recommended bait quantity was used. Following each round of capture, the hybridized library pools were amplified using the KAPA HotStart Library Amplification Kit (Roche) to obtain a minimum of 200 ng total DNA per library pool. After final quantification using the Kapa HiFi Library Quantification Kit (Roche) or the NEBNext Library Quant Kit for Illumina, the enriched pools were diluted to the recommended concentrations. Sequencing was performed on a MiniSeq platform (Illumina, USA) using the V3 chemistry (MiniSeq High output Kit for 75 or 150 cycles). For whole genome sequencing of MPXV from cell cultures, we generated libraries from isolates from two mangabey skin samples and from the squirrel lung using the Rapid-Barcoding Kit V14 (Oxford Nanopore Technologies, United Kingdom) and sequenced them directly on a PromethION 2 solo platform (Oxford Nanopore Technologies) using R10.4.1 PromethION flow cells. Basecaller v4.3.0 was set to super-accurate base calling v4.3.0, 400 bps.
**Sequencing data analyses.** Reads from different tissues of the same individual were merged to improve viral genome coverage. Raw sequencing reads were quality-filtered using trimmomatic v0.39³⁶ using the settings: LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 MINLEN:30. Filtered reads were then mapped to the most recent MPXV genome from TNP (GenBank accession number MN346702) using BWA MEM v0.7.17-r1188³⁷. Mapped reads were sorted and duplicates removed using SortSam and MarkDuplicates by Picard v2.13.3 (http://broadinstitute.github.io/picard/). In parallel, paired reads were mapped to the reference genome in Geneious Prime 2023.1.2 (https://www.geneious.com) using default settings to improve coverage of inverted terminal repeats regions of the MPXV genome. Consensus sequences were generated from the reference based mapping pipeline and the Geneious mapper and checked manually for concordance. Base calling was set to a minimum of 20 reads and 95% agreement. For samples where we obtained a shallow coverage, consensus sequences were called when two or more reads were present. Nearly complete viral genomes (excluding the ITRs) used for phylogenetic analyses were assembles from the reference based mapping. The complete mitochondrial genome of the squirrel was de-novo assembled from quality-filtered reads using SPAdes v3.13.0³⁸. Oxford Nanopore reads were quality trimmed using BBDUK with the following settings: qtrim=rl trimq=6 minlength=50 ordered=t qin=33 (BBMap - Bushnell B. - sourceforge.net/projects/bbmap) and de novo assembled using Flye v2.9.2³⁹ (flags --nano-hq; --genome-size 200k). The entire dataset was then re-mapped against the initially generated sequence through Minimap2 v2.17⁴⁰ (ONT mode; including secondary alignments; maximum secondary alignments per read = 5; minimum secondary to primary alignment ratio = 0.8).

**Phylogenetic analyses.** A dataset representing the current known MPXV clade IIa diversity was assembled from publicly available data on GenBank and GISAID⁴¹. For identical sequences originating from the same outbreak only one representative genome was selected. We also included partial genomes from Côte d'Ivoire and Liberia from 2024. After evaluating the Côte d'Ivoire sequences through Nextclade v3.12.036⁴² quality control (https://clades.nextstrain.org/), we identified three high quality genomes, which we included in our analysis. Furthermore, two genomes of lower quality were added due to their origin in geographic proximity to TNP. This dataset plus one representative MPXV genome per species from the TNP 2022/2023 outbreak (n=28) were aligned using MAFFT v7.505n⁴³. Gblocks v0.91b was run in SeaView v5 on stringent settings to not allow for many continuous nonconserved positions⁴⁴. Identical sites, ambiguities and gaps were removed in Geneious, so only variable sites were included in phylogenetic analysis (534 bases). The best fitting model of nucleotide evolution was identified using Modelfinder⁴⁵ with ascertainment bias correction as implemented in IQ-TREE v1.6.12⁴⁶,⁴⁷. A maximum likelihood phylogeny was reconstructed on IQ-TREE and branch robustness was assessed by ultrafast bootstrapp (1000 replicates)⁴⁸. The best-fitting root of the resulting tree was identified in TempEst v1.5.3⁴⁹ and further tree visualisation and editing was done in iTOL v7.1 (https://itol.embl.de/)⁵⁰.

**Diet analysis.** The mangabey's diet was analysed using a metabarcoding approach. The faecal samples used in this particular study (n=78) were collected just before the mpox outbreak started in the mangabey group (01.10.2022-30.12.2022). We used the Tagsteady protocol⁷. A first PCR assay targeting
a 130bp fragment of the 16S mitochondrial DNA was performed with tagged 16S mam1 (16Smam1 5'-CGGTTGGGGTGACCTCGGA-3') and 16S mam2 (5'-GCTGTTATCCCTAGGGTAACT-3') primers to identify each sample individually. This PCR was performed with the addition of human blocking primer (16Smam yap hum 5'-CGGTTGGGGCG ACCTCGGAGCAGAACCC-3') to reduce the amplification of contaminant human DNA. A total volume of 25 ul was used for each reaction, which included 2 ul of DNA template. The cycling parameters were set as follows: 95°C for 10 min, 35 cycles of 95°C for 12 s, 59°C for 30 s, 70°C for 25 s, and 72°C for 7 min. Subsequently, we generated three pools comprising all positive samples together with the positive and negative controls from the same PCR plate (three plates with one replicate each; 262 amplicons in total). After end repair, we indexed the pools by ligation with Illumina full-length Y-adapters that carried dual matching indexes (P5-P7) ⁵¹. The indexed pools were sequenced on an Illumina iSeq 100 System. To analyse the resulting reads, we first assembled a reference database from the EMBL collection of vertebrate sequences (downloaded on July 10th, 2024) on which we performed an in-silico PCR with the OBItools (v3.0.1b21) *ecopcr* command, allowing up to three mismatches between the primer and the reference sequences. We sorted the reads generated from the diet analysis to their respective PCR replicate using their 5' nucleotide tags using OBItools and removed primer sequences. Paired-end reads were then merged using the OBItools *Illuminapairedend* command keeping only reads with >0.8 alignment quality score and a length >80bp. Sequence variants were then collapsed with the *obiclean* command, but retaining a count of their appearance in each PCR replicate. We then compared the resulting aligned reads with our reference database to try to assign taxons by using the OBItools *ecotag* command. To consider a wildlife species detection event genuine, we required that at least two of the three replicates contained at least two times the maximum number of reads assigned a taxonomy in the negative controls. We also used Geneious to competitively map the trimmed reads to the mitogenome we generated from the squirrel spleen, as well as a human (NC_012920), chimpanzee (KU308547), and mangabey (NC_028592) mitogenomes.

**Re-analyses of fly data.** To investigate the distribution of fire-footed rope squirrels and sooty mangabeys along a local ecological gradient, we reanalysed a recently published mammal metabarcoding dataset derived from fly DNA²⁷. To do this, we applied the same bioinformatics approaches used for faecal diet analyses to the 100 datasets (25 from the forest, 50 from the edge, 25 from villages) produced by this study (doi: 10.5281/zenodo.7688127).

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## Figures
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

Fire-footed rope squirrel (*Funisciurus pyrropus*) range and map of the sooty mangabey (*Cercacebus atys*) territory in TNP. a Distribution of the fire-footed rope squirrel in Africa (light blue) according to IUCN redlist and the location of Taï National Park within Côte d'Ivoire for reference (green). bHome range of the habituated sooty mangabey group at TCP, approximated as the 95% minimum convex polygon (MCP) in black dashed line of the mangabey movements tracked over the period of one year (blue). The squirrel icon represents the location of the necropsy of the fire-footed rope squirrel which was positive for MPXV.
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

Different degrees of maculo-pustular rash associated to MPXV infection in three sooty mangabeys (*Cercacebus atys*). **a** Severe maculo-pustular rash (>20 lesions) spread all over the body. **b** Moderate maculo-pustular rash (5-20 lesions) localized on the head. **c** Mild maculo-pustular rash (1-5 lesions) localized on the neck. White arrows point at the observed skin lesions. Photo credit: TCP/Carme Riutord-Fe.
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

Detection of MPXV DNA and of fire-footed rope squirrel (*Funisciurus pyrropus*) DNA in sooty mangabey (*Cercacebus atys*) faeces. Each line represents a different individual. Filled circles represent virus-positive samples, empty circles represent virus-negative samples. The solid red circle indicates the detection of squirrel DNA in a sample that was also MPXV positive, while the unfilled red circle represents the detection of squirrel DNA in a sample that was MPXV negative. Vertical grey shadowing
indicates the period in which clinical signs were observed in the mangabey group. For each individual, the purple horizontal shadowing indicates the period during which clinical signs were observed.

<figure><img src="image_6.png" /><figcaption>**Figure 4**</figcaption></figure>

**Maximum likelihood phylogeny of clade IIa MPXVs.** Genomes retrieved from the sooty mangabey (*Cercacebus atys*) outbreak and the fire-footed rope squirrel (*Funisciurus pyrropus*) are shown in blue. The scale bar represents substitutions per variable site. Bootstrap support of the inner branches is indicated by colour (grey is < 0.90; black is ≥ 0.90).
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>

Sooty mangabey (*Cercacebus atys*) eating a fire-footed rope squirrel (*Funisciurus pyrropus*) in TNP. This adult female was observed eating a squirrel on December 9th 2014. Photo credit: TCP/Alexander Mielke

**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* RiutordExtendedDataFigure1.jpg

* RiutordExtendedDataFigure2.jpg

* RiutordExtendedDataFigure3.jpg

* RiutordExtendedDataFigure4.jpg

* RiutordSupplementaryInformationTables16.xlsx

* SupplementaryInformationGuide.docx

* RiutordSupplementaryVideo1.mp4
* RiutordSupplementaryVideo2 mov