Search of the animal reservoir of monkeypox virus based on museum specimens

Mpox is an emerging disease characterized clinically by a cutaneous rash associated with fever and lymphadenopathies. This disease in its classic African form usually follows a self-limiting course, except in malnourished children suffering from bacterial co-infection and also in some HIV infected patients. It is historically endemic in the forested areas of West and Central Africa. In 2022-2023, it has been epidemic with global world distribution through a sexual-transmitted dissemination mainly in men having sex with men. Such a pandemic has affected nearly 100,000 people with around 150 deaths. Currently, there is a significant increase in cases in Central Africa, particularly in the Democratic Republic of Congo with an epidemic in the east of the country and neighboring countries in the context of predominantly heterosexual human-to-human transmissions.Mpox is a zoonosis of African origin caused by the monkeypox virus (MPXV), a Orthopoxvirus. Despite many studies, its animal reservoir remains to be identified. However, several lines of evidence, including our recent study, based on ecological niches, point towards rodents, especially arborical squirrel species, such as Funisciurus anerythrus, living in African rainforests of West and Central Africa. Our hypothesis is that this African tree squirrel or a related species is the, or one of the animal reservoirs of MPXV. Our aims are to identify which rodent species is/are involved as reservoir host(s), to characterize its/their evolutionary history based on genetic data, to make comparison with the spatio-temporal evolution of MPXVs of zoonotic origins, and to detect and characterize the monkeypox virus in museum specimens collected in both Central and West Africa for years or decades. To conduct this project, we need to compare the phylogeography of several squirrel species with that of MPXV. For this, it is necessary to generate DNA sequences on georeferenced squirrels collected in both West and Central African rainforests. For this, we have already obtained a large series of DNA from these animals, using biopsies taken on specimens housed in museum collections (MNHN in Paris, RMCA in Tervuren, etc.). The "museomic" approach has two advantages for our study: it avoids any field mission to collect animals in the forests of Africa, which means no environmental impact, and it allows us to test the presence of the Monkeypox virus in specimens collected in the 19th and 20th centuries, which could be particularly useful for dating the past diversification of the virus. Our preliminary results indicate that DNA extracts from museum specimens can be amplified by PCR and sequenced by NGS generating genomic DNA of the hosts as well as of MPXV. The different steps of our study are : a) to sample additional squirrel specimens from museum collections to ideally have a series from most of the countries in West and Central Africa, b) to determine the quality of DNAs in order to select those to be used for PCR, exon capture and NGS, c) to assemble high-quality sequences from such specimens, d) to infer the phylogeography of several squirrel species distributed in tropical Africa, e) to make comparison with the phylogeography of monkeypox viruses of zoonotic origin both in terms of geographic patterns and divergence time estimates, f) to detect and characterize monkeypox viruses present in squirrel specimens in collections, g) to include these MPVX sequences of the 19th and 20th centuries in phylogenetic analyses in order to better estimate the molecular timescale of MPXV evolution. Based on all these data, we believe strongly that our experimental approach, combined with our expertise in mammalogy, evolutionary phylogeny and molecular virology, will enable us to respond rapidly to our working hypotheses concerning the animal reservoir of the monkeypox virus.