A One health study of Monkeypox: Human infection, animal reservoir, disease ecology and diagnostic tools – AFRIPOX

Through the study of past and current epidemics, we document the epidemic potential of the disease and the risk factors associated with zoonotic and human-to-human transmission, notably through a case-control study in Lobaye.

Ethnohistorical and anthropological approaches allow us to draw on local knowledge about the disease and the ecological and social changes that may have led to its emergence.

The search for the animal reservoir is done through animal captures around human epidemics, but also through the search for viral DNA in historical specimens from museum collections and through the comparison of the ecological niche of the virus and mammals suspected of being reservoirs.

The ecological approach developed allows us to determine the ecological factors associated with the emergence of monkeypox in central Africa and the potential geographic extent of monkeypox infection in this region.

The different virological approaches implemented in the project aim to improve the field diagnostic capabilities of monkeypox in CAR, to identify and respond rapidly to outbreaks.
New molecular field diagnostic tests will be developed and tested to rapidly discriminate between monkeypox and varicella.
More specific serological tests will be developed to obtain a reliable serological diagnosis.
Rapid sequencing methods will also be implemented to reinforce the current capabilities of the iPasteur Institute of Bangui.

Epidemiology
The last epidemic investigation in Lobaye in October 2019 highlighted the occurrence of epidemics in the context of seasonal collection activities in forest areas.
Analysis of retrospective national surveillance data provided a comprehensive epidemiological and clinical overview of the natural history of monkeypox in CAR between 1983 and 2021.

Anthropology
Development of tools

Zoology
1. Analysis of ecological niches of animal reservoirs likely to harbor Monkeypox virus.
To identify potential animal reservoirs of Monkeypox, the ecological niche of the virus was compared to all mammals in sub-Saharan Africa.

2. Phylogenetic and phylogeographic analyses of squirrels.
The genera Funisciurus and Heliosciurus are considered the most likely animal reservoirs for Monkeypox. We initiated a phylogenetic study of the Protoxerini. From 103 museum samples collected from 21 different species, we have so far obtained 94 sequences of the 1st subunit gene of cytochrome c oxidase. Our preliminary phylogeny revealed many taxonomic problems requiring a study of morphological characters to realize a determination key.

3. Research of monkeypox virus in squirrels from MNHN collections
We initiated a study to search for monkeypox DNA from skin samples collected from 110 specimens of eight species of Funisciurus (from 1891 to 1996), to test for the presence of the monkeypox virus.

Ecology
Creation of an ecological data atlas to link epidemic data with environmental contextual data.

Virology
1/Study and implementation of a diversification of sequencing approaches for the monkeypox virus
The use of the MinION allowed the improvement of the expected sequence at the extremities. A new targeted enrichment approach based on microfluidic technology has been initiated.

2/Improvement of the discriminatory capacities of the serological tests between the different Orthopoxviruses by studying the complete peptidome of the virus surface proteins to identify the specific antigens.
A total of 24 surface proteins were selected from the MPXV proteome (191 proteins) for the development of a multiplexed serological test. These transformed bacteria will be used to produce a library of bacteriophages expressing viral peptides on their surface, and thus to capture specific serum antibodies and identify the serological profiles of patients infected by MPXV.

3/Field molecular diagnosis using lyophilized reagents through 4 rapid isothermal amplification tests allowing to differentiate MPXV infections from Varicella-Zoster virus infections, and to identify MPXV lineage in infected individuals.

The interest of this project lies in a preventive management of these potential emergences in a generic way by understanding their epidemiology and the factors involved in their emergence, and in the strengthening of the health and surveillance systems of the countries concerned.

The identification of risk factors for zoonotic or environmental and human-to-human transmission will be a major advance in the understanding of this disease and its prevention.
The determination of the transmissibility of monkeypox in a population with decreasing immunological protection against Orthopoxviruses will allow a better understanding of the participation of the decrease of the post-vaccination anti-variola immunity in the increase of the disease incidence.

The qualitative approach brought by ethnohistorical and anthropological surveys will resituate monkeypox in these social and ecological contexts of emergence, through the knowledge of the local populations concerned.

The identification of the ecological characteristics of the areas affected by the disease will allow us to highlight the importance of ecological factors and recent environmental modifications in the increase of the incidence of this disease and to attempt to prevent it through land-use planning policies.

The different virological techniques developed during this project will allow to reinforce the rapid field diagnosis of monkeypox.
Improved sequencing capabilities will lead to a better understanding of the links between different human and animal cases and between epidemics, and will clarify the circulation of the virus between human and animal populations.

1. Berthet N, Descorps-Declère S, Besombes C, et al. Genomic history of human monkey pox infections in the Central African Republic between 2001 and 2018. Sci Rep. 2021;11(1):13085. Published 2021 Jun 22. doi:10.1038/s41598-021-92315-8

2. Mathias Vandenbogaert, Aurélia Kwasiborski, Ella Gonofio, Ste´phane Descorps-Decle`re, Benjamin Selekon, Andriniaina Andy Nkili Meyong, Rita Sem Ouilibona, Antoine Gessain, Jean-Claude Manuguerra, Valérie Caro, Emmanuel Nakoune2, Nicolas Berthet. Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic. In revision.

3. The natural history of the monkeypox virus in the Central African Republic, 1983-2021. Submission ongoing

4. Possible link between monkeypox virus outbreaks and edible catterpillar gathering in the Central African Republic, 2019. Submission ongoing

No patent.

Monkeypox, an emerging Orthopoxvirus with a similar disease presentation to smallpox, is a zoonotic virus which can spread from person to person. Although to date, monkeypox events have erupted in West and Central African rainforests, their frequency, size, and geographic scope have expanded substantially in recent years. Imported cases have been detected in multiple locations, including Europe. Yet many aspects of this emerging infectious disease remain unclear, including its animal reservoir, its risk factors for zoonotic and interhuman transmission and ecological characteristics that may facilitate monkeypox emergence. As a result, the 2018 WHO Research & Development Blueprint designated monkeypox as an emerging disease requiring “accelerated research & development and public health action”.

The AFRIPOX collaboration will mobilize an international, multidisciplinary One Health partnership spanning epidemiology, anthropology, zoology, environmental ecology, virology and mathematical modelling to tackle four research objectives investigating monkeypox at the human-animal-ecosystem interface:
- Identify the animal reservoir and secondary hosts of monkeypox in areas of Central Africa where monkeypox is known to circulate
- Identify risk factors for zoonotic and human-to-human monkeypox transmission during outbreaks using quantitative and qualitative methods; determine interhuman transmissibility of monkeypox and its epidemic potential
- Understand differences in viral strains circulating in animals and human populations; strengthen diagnostic and response capacities, develop field diagnostic and next generation sequencing capacities for monkeypox in areas of Central Africa where the virus is known to circulate
- Determine ecological factors associated with monkeypox emergence in Central Africa and its potential geographic scope in this region

The partners’ extensive field experience and knowledge, their internationally recognised expertise, and their existing collaborations will ensure achievement of these objectives. Systematic outbreak investigation and response will improve outbreak control and identify risk factors for spillover and for human-to-human transmission. Studies of past and on-going outbreaks will yield deeper understanding of recent trends in the epidemic potential of the disease and strengthen infection prevention and control measures.
An ethno-historical approach will excavate local knowledge about monkeypox and about the ecological and animal behavioural changes that precede or accompany outbreaks; such insights can point to new, unexplored investigations of ecological/zoological dynamics of monkeypox emergence. Investigation of local understandings of human transmission will permit the development of locally acceptable risk communications and prevention measures.
The virology work package will conduct phylogenetic analysis to compare human and animal viral strains; it will also improve diagnostic capacities by developing a specific serological assay and field diagnostic test. With strengthened surveillance and field-adapted methods, our study will reduce the time to response, critical for controlling monkeypox and reducing the importation risk, as well as for containing other viral emergence.
Understanding the animal species and environmental characteristics which contribute to the emergence of monkeypox will help to pinpoint zones at risk of zoonotic spillover and enhance outbreak preparedness activities there.
Through its investigation of monkeypox, AFRIPOX will set the critical foundations for understanding and controlling a global infectious threat. Our multi-disciplinary collaboration will address research about monkeypox and preparedness to outbreaks: it will develop crucial scientific knowledge, identify appropriate response, and strengthen Central African Republic capacity to manage this and other emerging zoonotic diseases, beyond the duration of the project.