Human monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus in the Poxviridae family. The clinical presentation generally resembles smallpox: after a mean incubation time of 8 days (4-14 days), most patients first experience fever and a few days later a characteristic rash1,2. Case fatality rate is usually low in adults, but can reach well over 10% in children. The virus naturally occurs in Africa where two genetic clades have been discerned: a West African clade (MPXV-WA) that is only found in countries situated west of Cameroon, and a Congo Basin (MPXV-CB) clade only observed in Central Africa1–3. While the MPXV-CB was generally considered to be more virulent and transmissible in humans (based on a low sample size)4, the recent Nigerian outbreak (2017-2018) provided evidence of increased virulence of MPXV-WA5. The increasing incidence and human-to-human transmission for both strains in endemic areas highlight the need for a better understanding of the eco-epidemiological drivers of the disease6.
The presumptive animal source of primary human infections remains unidentified and the virus’ natural reservoir cryptic7. MPXV has only been isolated from three African endemic wild mammals: in the Democratic Republic of the Congo (DRC) from a Thomas’s rope squirrel (Funisciurus anerythrus), and in the Taï National Park in Côte d’Ivoire from a sooty mangabey (Cercocebus atys atys) and western chimpanzees (Pan troglodytes verus)8–10. Whereas primates are assumed to be incidental hosts, different species of rodents are considered to be potential reservoirs of the virus (such as Cricetomys, Graphiurus, Funisciurus and Heliosciurus sp). However, empirical data to support these claims is scarce and mainly based on serological data or DNA amplicons without confirmation by sequencing11–15.
Since 2010 we have been monitoring the biodiversity of small mammals and their pathogens in several areas in the Ituri and Tshopo provinces of the DRC. A subset (n=256) of samples (random selection of samples that were completely submerged in ethanol, Supplementary excel file: ‘small_mammals_MPXV’), including tissues collected from rodents (n=97), shrews (n=93) and bats (n=66) over a period of six years, was tested for the presence of MPXV DNA using two PCR assays targeting different viral genes (P4A and haemagglutinin) and positives were confirmed by Sanger sequencing16,17. Tissues from individuals representing four rodent species (Funisciurus anerythrus, Funisciurus bayonii, Stochomys longicaudatus, Cricetomys sp. 2 sensu Olayemi et al. 2012) and one shrew (Crocidura littoralis) contained MPXV DNA. By applying in-solution hybridization capture and high-throughput sequencing10, we successfully retrieved two complete MPXV genomes from F. anerythrus and C. littoralis, and one partial genome (59%) from F. bayonii. Therefore, MPXV appears to naturally circulate in multiple, distantly-related rodent species from at least three different families (Sciuridae, Muridae and Nesomyidae), as well as in shrews (order Eulipotyphla). While some of these detection events may reflect dead-end infections, they are compatible with the hypothesis that MPXV has a broad host range and possibly a complex multi-host ecology18. The ability to infect multiple host species is also known for other OPXVs, including the better-studied cowpox virus (CPXV) which infects at least three rodent species (field voles, bank voles and wood mice) in the UK, sometimes leading to spillovers into bovids14. Because OPXVs are relatively stable in the environment and able to infect susceptible hosts via multiple routes (respiratory, mucosal and parenteral), cross-species transmission of OPXVs is likely facilitated by indirect transmission and might happen frequently between species18.
Although MPXV is able to infect multiple hosts, it does not necessarily imply that all hosts contribute to viral persistence. In fact, it is often suggested that the majority of transmission events and pathogen persistence is associated to a key host species19. Consequently, much of the apparent complexity of such multi-host systems could be simplified by focussing on one or two key hosts within the community. The key position of these hosts may be the outcome of separate processes such as infection prevalence, population density and transmissibility19. For example, while CPXV can be maintained in wood mice populations in the absence of other mammal species (potentially due to their high population abundance), its transmission in bank voles populations largely depends on the presence of wood mice or field voles in the wildlife community20. It is possible that the maintenance of MPXV also depends on one or two primary animal reservoirs and that other species serve as auxiliary hosts. The results reported here adds to the limited evidence pointing at Funisciurus squirrels as potential primary reservoirs for MPXV. Squirrels from this genus show the highest seroprevalence of all rodents and they have allowed for the isolation of the virus and now for its whole genome sequencing from multiple individuals11,12,15. Within this genus, F. anerythrus maybe sticks out as a particularly plausible primary host due to its social behaviour (more interaction with conspecifics) and higher densities than any other species of African tree squirrel, both characteristics facilitating viral intra-species transmission21.
Nevertheless, the fact that MPXV was first isolated from a Funisciurus squirrel might have biased our view on MPXV ecology, as most follow-up studies targeted squirrels and potentially neglected other animal orders, such as shrews or bats. Similarly, the multimammate mouse was for a long-time assigned to be the sole Lassa arenavirus (LASV) reservoir, as the virus was first isolated from this rodent species which is highly abundant around houses in areas where the disease is endemic. However, recent studies also detected LASV in rodent communities even in the absence of the multimammate mouse22. This suggests that the concept of a ‘primary reservoir’ might often depend on the context. For example, it might be that MPXV transmission is maintained by Funisciurus squirrels in areas where these rodents are abundant, while other species are needed for viral persistence in areas where Funisciurus squirrels are less abundant, or absent. Additional field studies targeting a larger variety of mammals and following populations over time are necessary to confirm the hypothesis that Funisciurus squirrels are indeed the primary MPXV reservoirs, or to support the hypothesis of ‘combined maintenance’ in the wildlife community.
Maximum likelihood (ML) and Bayesian phylogenetic analyses revealed that the MPXVs found in the red squirrel (F. anerythrus) and shrew are closely related to each other and fall within the diversity of MPVX strains found in humans in Central Africa (Fig 1), with the time to the most recent common ancestor (tMRCA) with the closest published genomes dated to 1959. Similarly, ML analyses performed on the partial MPXV genome found in the F. bayonii showed that it clusters with other MPXV-CB strains (Supplementary Fig. 1). The presence of the new animal-derived sequences provided an opportunity to reassess the standing hypothesis that MPXV diversification essentially reflects ancient vicariance events caused by climate-induced forest-habitat contractions, such as the opening of the Dahomey gap (4,500BP) or the Last Glacial Maximum (21,000BP)2. We found, however, that such diversification was too recent to be compatible with these events, with the MPXV tree root being about 600 years-old in the best model. However, given the relatively modest divergence between MPXV-WA and MPXV-CB, we do not expect this would affect the estimated evolutionary time scale of MPXV to such an extent that it would become compatible with key climate factors (4,000-25,0000BP) as proposed in Nakazawa et al2. Rather, we consider that it is more likely that the geographical isolation of the two clades has occurred more recently (e.g. during the Medieval Warm Period; 700-1,100BP)23 or that alternative evolutionary mechanisms were at play. The WA and CB MPXV viruses may for example circulate in distinct hosts communities, with MPXV-CB depending on hosts strictly endemic to Central Africa for its long-term persistence.
In conclusion, the presence of MPXV DNA in diverse forest dwelling rodents and shrews sheds a new light on the ecology of MPXVs. This finding calls for increased surveillance in a wider range of African mammals to reveal the host range and distribution of MPXV, and possibly to discern whether different animal groups host distinct MPXV strains with distinct virulence.