##Limited Ebola Virus Exchange Across the Sierra Leonean Border

A previous study of EBOV Makona sequences elucidated viral transmission and evolution during the early stages of the outbreak in Sierra Leone \citep{Gire_2014}, from late May to early June, 2014. The first reported EVD cases in Sierra Leone stemmed from two genetically distinct EBOV Makona lineages, believed to have been introduced from Guinea. One of these lineages (SL1) was more closely related to the then-available three Guinean genomes (2-5 mutations) than the second lineage (SL2), which was characterized by 4 additional mutations. This finding suggested that SL2 had evolved from SL1 some months before it was observed in Sierra Leone. A third lineage (SL3), derived from SL2, emerged in mid-June 2014. SL3 differs from SL2 by a single mutation at position 10,218, first found as an intrahost variant (polymorphism within one individual) at a low frequency. SL3 became the most prevalent lineage in Sierra Leone during the first three weeks of the outbreak there, with SL1 disappearing soon after the appearance of SL3. The SL3-defining mutation is epidemiologically important, as it is the first commonly circulating mutation observed to arise within Sierra Leone's borders.

As the epidemic developed within Sierra Leone, the SL3 lineage continued to dominate the viral population within the country, with no evidence for additional imported EBOV lineages. In our data set, 97% of the genomes belong to SL3, and the remainder to SL2 (Figure 1A). These results link all Sierra Leonean EVD cases to the initial introduction of EBOV into Sierra Leone, and they provide further evidence that all EVD cases during this outbreak arose from human-to-human transmission rather than from further zoonotic introductions from the unknown EBOV reservoir. This means that no newly imported viral diversity was detected after the initial introduction \citep{Gire_2014}; all newly sampled viruses likely descended from those sequenced in the initial weeks of the outbreak. The genetic similarity of these viruses suggests importation from other countries was minimal, although we cannot definitively rule out a re-introduction from elsewhere for the SL2 viruses (3%) in our data set.

Similarly, publicly available EBOV genomes from this outbreak can shed light on exportation of EBOV from Sierra Leone into other countries. All published genomes from elsewhere, including 26 from Liberia and 4 from Mali, lack the Sierra Leone-defining SL3 mutation (Figure 1B; Experimental Procedures). Given that 97% of Sierra Leonean EBOV sequences are SL3, extensive exportation would result in the spread of SL3 EBOV genomes, a spread that is not seen in the limited samples available to date. At least in Sierra Leone, and with the exception of events at the onset of the epidemic, transmission has likely been primarily within national borders (Figure S2, Experimental Procedures), rather than by free interchange with neighboring countries.