Our phylogenetic analysis strongly supports the existence of distinct clades from the Congo Basin and western African, and all subclades were well supported. Sudan isolates 1 and 2 are imbedded within the Congo Basin MPXV clade, specifically within a northern DRC subclade. However, we did not have samples from the northernmost Congo Basin forest, which is closer to Sudan. In comparison of the position of the isolates from the 2005 Sudan outbreak with that of other isolates from Congo Basin during 1986–2003, the former cannot be distinguished as a new strain of MPXV on the basis of these phylogenetic analyses. Furthermore, the isolates most closely related to the Sudan isolate are from Yandongi and Mindembo DRC, suggesting that the virus obtained during the 2005 outbreak probably originated from northern DRC ().
The Sudan isolates uniquely duplicate a 10.8-kb sequence that represents a single mutation event. Given the overall similarity to the Congo Basin isolates, this single duplication is not considered sufficient evidence to suggest an independent evolutionary trajectory. Formenty et al. (5
) proposed that the Sudan virus was novel among Congo Basin isolates because of this large duplication of genetic information not seen in other monkeypox viruses sequenced to date. The changes seen in the 4 regions between the 2 sequenced Sudan isolates were not seen in the sequenced monkeypox isolates from the 2003 US outbreak of monkeypox. Further genetic analyses could help clarify epidemiologic details through examination of genetic variations accumulated during a single outbreak, but these analyses are beyond the scope of the current study.
The long-term maintenance and transmission of a virus in wildlife would presumably require genotypic adaptations to susceptible hosts, which in turn are adapted to the environmental characteristics of a particular region. Although there are some differences between the Sudan MPXV and other Congo Basin viruses, these differences are well within the limits of variation seen within the Congo Basin clade. The 2 recognized MPXV clades (West Africa and Congo Basin clades) have been described in areas in which the dominant ecosystem is tropical rainforest. Although there are slight habitat differences between the MPXV ranges within western Africa and the Congo Basin, the grassland environmental characteristics and habitat descriptions at the outbreak localities in Sudan are dramatically different and do not fit the expected suitable environmental conditions on the basis of current knowledge of the 2 MPXV clades. This observation is supported by the ENMs (), in which the 2005 outbreak localities are not identified as suitable for MPXV transmission and life cycle maintenance.
The 2 possible explanations for the source of the virus that caused the 2005 monkeypox outbreak in Sudan are 1) the existence of conditions permitting the long-term maintenance of MPXV in wildlife within the area where this outbreak occurred and transmission of the virus from reservoir hosts into humans; and 2) the importation of MPXV into the outbreak area by an infected human or animal. The first hypothesis cannot be supported by the results from ENMs and the criteria of ecological niche conservatism between genetically differentiated taxa, which demonstrate that genetic differentiation occurs faster than ecological differentiation (38
). On the basis of the ecological differences described in this study, we would expect indigenous isolates from Sudan to have high genetic differentiation when compared with isolates from MPXV clades found in West Africa and Congo Basin. Our genetic analysis, however, groups Sudan isolates 1 and 2 within the Congo basin clade; therefore, we consider the hypothesis of an indigenously acquired infection to be unlikely.
Genetic similarity between isolates from DRC and the sample obtained from the 2005 Sudan MPXV outbreak support the second hypothesis, importation of the virus. Additionally, human nomadic events, displacement and repatriation of Sudanese residents, were characteristic of southern Sudan and northeastern DRC during the time of the outbreak (39
). Members of the Lord’s Resistance Army fled from Sudan into the Oriental Province of DRC persecuted by the Uganda People’s Defense Forces during September 2005 and were reported to have left the area by October (http://www.irinnews.org/report.aspx?reportid
= 57083). In January of that year, the Comprehensive Peace Agreement was signed, representing the end of a civil war that started in 1983 between northern and southern Sudan and giving autonomy to southern Sudan until 2011, when a referendum on independence was held, resulting in the recognition of South Sudan as a country. The end of the civil strife in Sudan precipitated the return of refugees who had sought assistance in neighboring countries (including DRC). Some sources reported the spontaneous return of thousands of persons from southern Sudan to their homeland in 2005 (http://reliefweb.int/node/198511
). Whether the movements of these persons are linked to the cases of monkeypox in Sudan may never be known with certainty, but the circumstances could have facilitated the importation of the disease by translocation of an infected animal or person from DRC.
An MPXV endemic to Sudan should reflect its adaptation to different hosts and ecological environments with respect to the currently known areas where the disease is endemic in the form of genetic divergence. Given the dramatic difference in ecology between the region surrounding Nuria, Sudan, and historic points of MPXV occurrence, the genomic comparisons between the Sudan isolate and other strains of MPXV would be expected to reveal genetic divergence as great as or even exceeding that observed between the 2 currently recognized MPXV clades in western and central Africa. However, our data indicate that the Sudan MPXV isolates and an MPXV isolate from Yandongi in north-central DRC are genetically similar to each other, even though they were collected 19 years apart from ecologically disparate and geographically discrete locations. For reference, these isolates from Yandongi and Sudan were more genetically similar to each other than the Yandongi and Mindembo DRC isolates (), that were the geographically and temporally closest isolates studied.
Further serologic surveys of human, animal, or both populations in Sudan could provide useful evidence in the investigations of the origin of the virus that caused this outbreak in Sudan. In addition, increased disease surveillance, ecological studies, and further characterization of the variability within and between clades will improve our understanding of the natural history of MPXV. Further epidemiologic studies to identify the sources and potential risks of MPXV infection in localities inside and outside the areas in which the disease is known to occur are clearly warranted.