LETTER

doi:10.1038/nature14490

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone Yi-Gang Tong1*, Wei-Feng Shi2*, Di Liu3*, Jun Qian5*, Long Liang1*, Xiao-Chen Bo4*, Jun Liu6*, Hong-Guang Ren1*, Hang Fan1*, Ming Ni4*, Yang Sun5*, Yuan Jin1, Yue Teng1, Zhen Li4, David Kargbo7, Foday Dafae7, Alex Kanu8, Cheng-Chao Chen9, Zhi-Heng Lan9, Hui Jiang9, Yang Luo10, Hui-Jun Lu5, Xiao-Guang Zhang6, Fan Yang11, Yi Hu1, Yu-Xi Cao6, Yong-Qiang Deng1, Hao-Xiang Su11, Yu Sun1, Wen-Sen Liu5, Zhuang Wang1, Cheng-Yu Wang5, Zhao-Yang Bu5, Zhen-Dong Guo5, Liu-Bo Zhang12, Wei-Min Nie13, Chang-Qing Bai14, Chun-Hua Sun1, Xiao-Ping An1, Pei-Song Xu4, Xiang-Li-Lan Zhang1, Yong Huang1, Zhi-Qiang Mi1, Dong Yu1, Hong-Wu Yao1, Yong Feng15, Zhi-Ping Xia5, Xue-Xing Zheng5, Song-Tao Yang5, Bing Lu1, Jia-Fu Jiang1, Brima Kargbo7, Fu-Chu He16, George F. Gao3,6,17, Wu-Chun Cao1 & The China Mobile Laboratory Testing Team in Sierra Leone{

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths1,2. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir3 followed by sustained human-to-human infections4. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics5,6. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase7. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 3 1023 substitutions per site per year (95% highest posterior density interval, 1.04 3 1023 to 1.41 3 1023 substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics. A large-scale Ebola viral disease (EVD) outbreak has been ongoing in Western Africa for nearly a year, with more than 23,000 reported cases1. Previous findings have shown that the causative agent is a novel Ebola virus (EBOV)2. Among the three West African countries with widespread and intense EBOV transmission, Sierra Leone reported the largest number of confirmed cases, approximately 58% of the total confirmed EBOV infection cases. To help Sierra Leone fight against EVD, the Chinese government dispatched the China Mobile Laboratory Testing Team (CMLTT) in September upon request of the Sierra Leone government. The CMLTT, equipped with medical experts who specialize in laboratory testing, epidemiology, and running a holding and treatment centre, has kept working at the Sierra Leone-China Friendship Hospital at Jui Town (represented as a red

star in Fig. 1a) of Western Area, approximately 30 km southeast of Freetown, the capital city of Sierra Leone. All the activities of the CMLTT were coordinated by the Emergency Operations Center jointly established by the Ministry of Health and Sanitation of Sierra Leone and the World Health Organization (WHO). To fight against this novel EBOV, Gire and colleagues systematically analysed 81 EBOV genomes from Guinea (n 5 3)2 and Sierra Leone (n 5 78)4 collected from the early stage of the 2014 EBOV outbreak, revealing the origin, transmission, and rapid accumulation of genetic variation of the 2014 EBOV. However, only a few additional full-length EBOV genome sequences were published since July 2014, when the outbreak entered a rapid growth phase driven by sustained human-tohuman transmission4. From 28 September to 11 November 2014, a total of 823 samples were tested to be EBOV-positive using reverse transcription-PCR (RT–PCR) by the CMLTT, among which 175 fulllength genomes were successfully sequenced with each from an individual EVD patient (Fig. 1a and Supplementary Table 1). These 175 samples were obtained from five severely stricken districts in Sierra Leone, including 47 from Western Urban, 67 from Western Rural, 47 from Port Loko, 5 from Kambia, and 9 from Bombali (Fig. 1a). In detail, approximately one fifth of the EBOV-positive samples for each region were sequenced, 19.5% for Western Urban, 21.2% for Western Rural, 22.1% for Port Loko, and 16.1% for Kambia. Regarding Bombali, 9 out of 17 (52.9%) strains were sequenced. Therefore, our sequenced genomes were roughly proportional to the prevalence in different regions. Phylogenetic analysis of all available full-length EBOV genome sequences from Sierra Leone (n 5 253) and Guinea (n 5 3) from 2014 was performed using MrBayes8 in which the three Guinean strains were designated as root4,9. Our phylogenetic analysis showed that the 2014 EBOV increased in diversity at least through October after its initial introduction into Sierra Leone (Fig. 1b and Extended Data Fig. 1). Apart from the previously described lineages SL1 and SL24, the SL3 lineage has evolved into two major lineages, SL3.1 and SL3.2 in June in eastern Sierra Leone, both of which were then transmitted to western Sierra Leone. The majority of the EBOV collected from late September to mid-November fell into lineage SL3.2, with a few belonging to lineage SL3.1. However, none of them belonged to lineages SL1 and SL2. In particular, the EBOV sequenced by us could

1

State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, China. 2Institute of Pathogen Biology, Taishan Medical College, Taian 271000, China. 3Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. 4Beijing Key Laboratory of New Molecular Diagnostics Technology, Beijing 100850, China. 5Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun 130122, China. 6Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 7Sierra Leone Ministry of Health and Sanitation, Freetown, Sierra Leone. 8Sierra Leone-China Friendship Hospital, Freetown, Sierra Leone. 9BGI-Shenzhen, Shenzhen 518083, China. 10Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK. 11 Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China. 12Institute of Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention, Beijing 100021, China. 13The No. 302 Hospital, Beijing 100039, China. 14The No. 307 Hospital, Beijing 100071, China. 15Department of international cooperation, National Health and Family Planning Commission, Beijing 100044, China. 16State Key Laboratory of Proteomics, Beijing 102206, China. 17Chinese Center for Disease Control and Prevention, Beijing 102206, China. *These authors contributed equally to this work. {Lists of participants and their affiliations appear at the end of the paper 0 0 M O N T H 2 0 1 5 | VO L 0 0 0 | N AT U R E | 1 G2015

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RESEARCH LETTER b Guinea SL1

a

SL2

8.0 × 10–5

3.1.1

1.0

SL3.1

1.0

47/213

3.1.2

Port Loko 3.2.1

0.93 0.99 0.99

3.2.2

Guinea Freetown

Guinea

0/3

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Kambia Waterloo

0.99

9/17

Bombali 0/1

3.2.4

Kono

Tonkolili

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an Oce

^

ntic

67/316

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n Atla

Koinadugu

5/31

^

tic O

cea

n

47/241

3.2.3

1.0

0/1

Moyamba

Kailahun

Bo

1.0

Kenema Bonthe Pujehun

Figure 1 | Geographical distribution and phylogenetic analysis of the 2014 EBOV from Sierra Leone. a, Geographical distribution of the 823 EBOV positive samples and the 175 newly sequenced genomes (represented as blue dots). In the panel, main roads and waterways are showed as yellow lines and

be classified into seven novel independent sublineages based on the phylogenetic topology, two sublineages belonging to SL3.1 (SL3.1.1 and SL3.1.2) and five belonging to SL3.2 (SL3.2.1 to SL3.2.5) (Fig. 1b). Phylogenetic tree constructed using the maximum likelihood method showed a similar topology (Extended Data Fig. 2). Therefore, the 2014 EBOV has become highly diverse in its first year along with its spread in Sierra Leone. To explore the spatiotemporal relationships of the EBOV in western Sierra Leone, we performed a phylogeographic analysis using BEAST10 (Fig. 2 and Extended Data Fig. 3). In this analysis, only 22 out of the 78 sequences previously published by Gire et al. (ref. 4) were included in our analysis to reduce the computation load. To this end, we selected representative sequences from the previously described lineages GIN, SL1, SL2 and SL3, ensuring that there is at least one sequence for every sampling date. From a time point of view, all of the novel sublineages probably emerged before August (Fig. 2). In addition, multiple lineages were co-circulating in a single town/district. All of the seven sublineages were identified in Waterloo, indicating the highest phylogenetic diversity in this region. Viruses from Freetown belonged to six of the seven sublineages, with sublineage 3.2.3 undetected. Five novel sublineages have also been found in Maforki Chiefdom of Port Loko. The spatiotemporal linkage of our sequenced EBOV genomes is further shown in Fig. 3a. First, viruses from Freetown and Waterloo, the capital and the traffic hub, are estimated to be spatiotemporally related, as also observed in sublineages 3.1.1, 3.1.2 and 3.2.4 (Fig. 2), indicating that frequent transmission events might have occurred between the two regions. Second, this network reveals that viral transmission events have also occurred between the three major sites (Freetown, Waterloo and Maforki Chiefdom) and their surrounding regions. Third, our results also suggest spatiotemporal connections of EBOV between Waterloo and Port Loko, Kambia, and Bombali, respectively, as exemplified in sublineages 3.2.1 and 3.2.5 (Fig. 2). Based on the higher transmission rates of Waterloo, Freetown and Maforki Chiefdom, intensive EBOV surveillance in

3.2.5

Liberia

black dash lines, respectively. b, A Bayesian phylogenetic tree of the 2014 EBOV. The 175 newly sequenced viruses in this study are shown in colours, and others are shown in grey. The seven novel lineages designated in the present are highlighted. Posterior support for major nodes is shown.

0.74 1.0

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0.07 Locations of lineages Mar

Apr May Jun

Jul

Aug Sep Oct Nov Dec

Figure 2 | Phylogeographic reconstruction of the 2014 EBOV using BEAST. In the left panel, the novel 175 EBOV genome sequences were coloured by geographic regions. The transition of different colours represents a potential transmission event. In the right panel, the number of sequences from different geographic regions in each lineage is summarized.

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LETTER RESEARCH

Maforki Chiefdom Rest of Port Loko

Port Loko tern Urban Western

Freetown Rest of Western

b Posterior probability

Bombali

c

0.5 0.4

1.23 × 10–3

EBOV all in 2014 EBOV Mar–Jun 2014 (ref.4)

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Situation report Patient database

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Kambia

Number of cases

a

28 Jul–3 Aug

29 Sep–5 Oct

1 Dec–7 Dec

Weeks

Figure 3 | Reconstructed phylogeographic linkage, substitution rate, and effective population size of the 2014 EBOV in western Sierra Leone from September to November 2014. a, The phylogeographic linkage constructed using BEAST. Thickness of lines represents the relative transmission rate between two regions. The size of each node is proportional to the sum of the relative rates of the region with Bayes factor .3. b, Substitution rates of the

2014 EBOV. The red line represents the substitution rate estimated using all the 2014 EBOV samples. Estimations of Gire and colleagues were repeated by us and shown as the blue line. c, Gaussian Markov random field Bayesian skyride reconstruction of the 2014 EBOV. Bar chart, adapted from the World Health Organization website, shows the numbers of confirmed cases of EBOV infection and patients. Smooth black line shows the effective population size.

the three regions should be helpful for the prevention and control of the EVD outbreak in Western Sierra Leone. The substitution rate for all of the 2014 EBOV was estimated using BEAST to be 1.23 3 1023 substitutions per site per year (95%

highest posterior density interval, 1.04 3 1023 to 1.41 3 1023 substitutions per site per year) (Fig. 3b). Our estimate was similar to those between previous EBOV outbreaks, approximately 1.00 3 1023 substitutions per site per year4,11–14. This suggests that, over a longer

Normalized cov.

a 2.5 2.0 1.5 1.0 0.5 0

NP

b

VP35 VP40

GP

VP30 VP24

Figure 4 | Genomic variations of the 2014 EBOV. a, Sequence depth across sequenced genomes. The x axis represents the virus genome structure, and the y axis represents the normalized average depth. One unit equals approximately 1,400 coverage per site. The mean depth is shown using the red line and the standard deviation is shown in shade. b, Substitutions of the 2014 EBOV. Only positions with substitutions are shown. Different lineages are separated by lines. Different types of substitutions are indicated using different colours: cyan for synonymous (S), magenta for non-synonymous (NS), green for un-translated regions (UTR), and grey for intergenic regions (IG). c, All the serial T . C substitutions are found within a range less than 150 bp. Substitutions within coding regions are shown in codons.

L

GIN SL1 SL2

SL3 3.1.1 3.1.2 3.2.1 3.2.2 3.2.3

3.2.4

J0024, J0028

5604

5594 5595 5596

5587

5566

5553

9651 9652

9632

T T T T C C C C

9646

T>L

T T TT T T T TTT T C C CC C C C CCC C 9527

V>A

5519

V>A

T C

5522 5523

TTT CTC

9518

GTT GCT

5512

GT T GCC

J0169

Asn

9507

1387

1366

Leu

9504

Pro

8082

Pro

8065

8010 8011

Arg

J0127

1354

CGT CCT CCT CT T AAT CGC CCC CCC CTC AAC 8063

J0157, J0150

8019

c

1267

1258

3.2.5

T T TT C C CC

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RESEARCH LETTER time interval, EBOV is still undergoing evolution at a relatively constant rate. The estimated population size of the 2014 EBOV from Sierra Leone steadily increased from July to early October, and then entered a plateau period (Fig. 3c). This therefore implies that the effective population size of the 2014 EBOV became stable in October, which was also broadly consistent with the weekly change of numbers of confirmed EBOV infection cases and EVD patients in Sierra Leone (Fig. 3c)1. The doubling time estimated using BEAST was 22.1 days (95% confidence interval, 18.9–25.59 days), which was comparable to that calculated using the epidemiological data from Sierra Leone, with the mean value of 18.9 days. We then investigated the molecular characterization of the novel EBOV genome. Raw reads of each genome were mapped to the reference genome (KJ660346.2). The average normalized coverage was approximately 1,400-fold (Fig. 4a). 341 single nucleotide polymorphisms (SNPs) have been previously identified between the 2014 outbreak EBOV and previous EBOV4, and 440 SNPs were identified in our sequenced genomes. The substitutions in the 175 newly sequenced EBOV genomes were summarized among different lineages (Fig. 4b and Supplementary Table 2). Approximately a quarter of the identified substitutions were non-synonymous, and half of them were synonymous (Extended Data Fig. 5). Some of the SNPs were lineagespecific and could be used as markers to distinguish different lineages (Fig. 4b and Supplementary Table 2). For example, substitutions A7148G and A17445G were only found in sublineage 3.1.2, whereas sublineage 3.2.4 possessed a specific T5849C substitution. The T . C substitutions that occurred in the 39 UTR region of NP gene (at genome positions 3008 and 3011) were specific to sublineage 3.2.5. In particular, the T . C substitution at position 14019 occurred in all sequences of lineage 3.2, which was first described in this study. Moreover, seven previously reported substitutions (at positions 800, 1849, 6283, 8928, 10218, 15963, 17142)4 were always present in the novel lineages from June to November 2014 and became the dominant allele in the population, suggesting that they have been fixed. These substitutions included two non-synonymous substitutions (C800T in the NP gene and C6283T in the GP gene), four synonymous substitutions, and one in the non-coding regions. Interestingly, we observed several serial T . C substitutions in six newly sequenced EBOV genomes, which occurred within a genome region of 150 base pairs in length (Fig. 4c and Extended Data Fig. 4). The serial T . C substitutions were further confirmed by Sanger sequencing after PCR amplification (Extended Data Table 1). Such serial substitutions were found in four different regions of six strains belonging to three different lineages, two of which were in coding regions and the other two were in non-coding regions. However, the emergence mechanism of such serial T . C substitutions and their potential biological functions warrant further investigation. In summary, our findings highlighted the increasing genetic diversity and transmission dynamics of the 2014 EBOV, with an evolutionary rate estimated to be similar to that between previous EBOV outbreaks. This information provided an insight into the viral evolution and transmission dynamics, which would facilitate the prevention and control of EBOV in Sierra Leone and would also guide research on vaccines and therapeutic targets. Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper. Received 30 January; accepted 23 April 2015. Published online 13 May 2015. 1.

World Health Organization. Ebola response roadmap - Situation report. http:// www.who.int/csr/disease/ebola/situation-reports/en (accessed 1 April 2015).

2. 3. 4. 5.

6. 7.

8. 9.

10. 11. 12.

13. 14.

Baize, S et al. Emergence of Zaire Ebola virus disease in Guinea. N. Engl. J. Med. 371, 1418–1425 (2014). Leroy, E. M. et al. Fruit bats as reservoirs of Ebola virus. Nature 438, 575–576 (2005). Gire, S. K. et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science 345, 1369–1372 (2014). Kugelman, J. R. et al. Evaluation of the potential impact of Ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics. MBio 6, e02227–14 (2015). Feldmann, H. et al. Ebola virus: from discovery to vaccine. Nature Rev. Immunol 3, 677–685 (2003). WHO Ebola Response Team. Ebola virus disease in West Africa–the first 9 months of the epidemic and forward projections. N. Engl. J. Med. 371, 1481–1495 (2014). Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755 (2001). Dudas G., Rambaut A. Phylogenetic analysis of Guinea 2014 EBOV Ebolavirus outbreak. PLoS Curr. http://dx.doi.org/10.1371/currents.outbreaks.84eefe5ce43 ec9dc0bf0670f7b8b417d (2014). Drummond, A. J. &. Rambaut. A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214 (2007). Jenkins, G. M. et al. Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis. J. Mol. Evol. 54, 156–165 (2002). Calvignac-Spencer S., et al. Clock rooting further demonstrates that Guinea 2014 EBOV is a member of the Zaı¨re lineage. PLoS Curr. http://dx.doi.org/10.1371/ currents.outbreaks.c0e035c86d721668a6ad7353f7f6fe86 (2014). Carroll, S. A. et al. Molecular evolution of viruses of the family Filoviridae based on 97 whole-genome sequences. J. Virol. 87, 2608–2616 (2013). Li, Y. H. &. Chen. S. P. Evolutionary history of Ebola virus. Epidemiol. Infect. 142, 1138–1145 (2014).

Supplementary Information is available in the online version of the paper. Acknowledgements We thank P. Lemey and S. Ho for technical assistance. This work is partially supported by the special project of Ebola virus research from the President Foundation of Chinese Academy of Sciences. It was also supported by grants from the China Mega-Project on Infectious Disease Prevention (nos 2013ZX10004202-002, 2013ZX10004605), China Mega-Project on Major Drug Development (no. 2013ZX09304101) and the National Hi-Tech Research and Development (863) Program of China (nos 2014AA021402, 2014AA021501). We thank the government of Sierra Leone, the Sierra Leone Ministry of Health and Sanitation and the Chinese National Health and Family Planning Commission. We also thank the medical workers and volunteers in Sierra Leone. G.F.G. is a leading principal investigator of Innovative Research Group of the National Natural Science Foundation of China, NSFC) (grant no. 81321063). Author Contributions The manuscript was written by Y.-G.T., W.-F.S., D.L., G.F.G. and W.-C.C. Samples were collected by J.Q., D.K., F.D., A.K., B.K., Y.S., H.-J.L., X.-G.Z., F.Y., Y.H., Y.-X.C., Y.-Q.D., H.-X.S., Y.S., W.-S.L., Z.W., C.-Y.W., Z.-Y.B., Z.-D.G., L.-B.Z., W.-M.N., C.-Q.B., C.-H.S., Y.F., Z.-P.X., X.-X.Z., S.-T.Y. and B.L. Experiment and data analysis were performed by Y.-G.T., W.-F.S., D.L., H.F., M.N., H.-G.R., J.L., Y.J., Y.T., Z.L., C.-C.C., Z.-H.L., H.J., Y.L., X.-P.A., P.-S.X., X.-L.-L.Z., Y.H., Z.-Q.M., D.Y., H.-W.Y., J.-F.J., X.-C.B., L.L., F.-C.H. and W.-C.C. The study was designed by B.K., X.-C.B., L.L., J.Q., F.-C.H., G.F.G. and W.-C.C. Author Information The 175 newly sequenced genomes have been submitted to GenBank. The accession numbers are provided in Supplementary Table 1. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to W.-C.C. ([email protected]), G.F.G. ([email protected]) or F.-C.H. ([email protected]).

The China Mobile Laboratory Testing Team in Sierra Leone Yi-Gang Tong1, Jun Qian2, Yang Sun2, Hui-Jun Lu2, Xiao-Guang Zhang3, Fan Yang4, Yi Hu1, Yu-Xi Cao3, Yong-Qiang Deng1, Hao-Xiang Su4, Yu Sun1, Wen-Sen Liu2, Zhuang Wang1, Cheng-Yu Wang2, Zhao-Yang Bu2, Zhen-Dong Guo2, Liu-Bo Zhang5, Wei-Min Nie6, Chang-Qing Bai7, Chun-Hua Sun1, Yong Feng8, Jia-Fu Jiang1 & George F. Gao3,9,10 1 State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, China. 2Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun 130122, China. 3Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 4Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China. 5Institute of Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention, Beijing 100021, China. 6The No. 302 Hospital, Beijing 100039, China. 7The No. 307 Hospital, Beijing 100071, China. 8Department of international cooperation, National Health and Family Planning Commission, Beijing 100044, China. 9Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. 10Chinese Center for Disease Control and Prevention, Beijing 102206, China.

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LETTER RESEARCH METHODS Ethics statement. This work was conducted as part of the surveillance and public health response to contain the EVD outbreak in Sierra Leone. Blood samples from suspected individuals and oropharyngeal swab samples from corpses were collected for EVD testing and outbreak surveillance with a waiver to provide a written informed consent during the EVD outbreak under the agreement between the Sierra Leone government and Chinese government. The activities were coordinated by the Emergency Operations Centre in the charge of Sierra Leone Ministry of Health and Sanitation and WHO. All the information regarding individual persons has been anonymized in the report. Genome sequencing and assembly. RNA samples extracted from whole blood from 175 EVD patients were reverse transcribed to cDNA. PCR amplifications were performed with EBOV-specific primer pairs with overlaps. Amplicons from one patient were pooled for library preparation. Next generation sequencing (NGS) was performed using the BGISEQ-100 (Ion Proton) platform. All the sequenced reads were filtered to remove the low quality and short reads. The genome sequences of the viruses were assembled by mapping the filtered reads to the 2014 EBOV consensus sequence using Roche 454 Newbler version 2.9 (Roche), and the mutation site was manually checked with original sequencing data. Phylogenetic and phylogeographic reconstruction. All previously published EBOV genome sequences and our newly released 175 sequences were aligned using MAFFT v7.05815. Phylogenetic analyses were performed using MrBayes8 v3.2 (10 million generations) and RAxML v8.1.6 (1000 bootstrap replicates), with the GTR model of nucleotide substitution and c-distributed rates among sites. Phylogeographic reconstruction of the 2014 EBOV was estimated using BEAST v1.8.010, with a continuous time Markov Chain (CTMC) over discrete sampling locations. The 175 newly sequenced samples in this paper were grouped into 7 regions (Waterloo, Freetown, Rest of Western, Maforki Chiefdom, Rest of Port Loko, Bombali and Kambia). Bayesian Markov chain Monte Carlo analysis was run for 100 million steps, 10% of which were removed as burn-in and sampled

G2015

every 10,000 steps. Bayes factor tests were performed to provide statistical support for potential transmission routes between different geographic locations using SPREAD v1.0.616. Bayes factors for rates were derived from a Bayesian stochastic search variable selection procedure. The phylogeographic linkage was constructed by routes with Bayes factor values .3. Substitution rates and population dynamics. The substitution rates were estimated using Bayesian Markov chain Monte Carlo (MCMC) as implemented in BEAST v1.8.0. In this analysis, two data sets were compiled, with one including all the 2014 EBOV sequences and the other including sequences from September to November, 2014. We performed two independent runs for 100 million generations, sampling every 10,000 steps. In addition, to accurately estimate the substitution rate, we repeated this analysis using a previously described data set using the same parameters. Population dynamics of the 2014 EBOV in Sierra Leone was estimated using a flexible non-parametric Bayesian skyride model17 incorporated in BEAST v1.8.0, with the HKY1C model and a strict molecular clock. Molecular characterizations of the 2014 EBOV. SNPs were called directly from the sequence alignment using the CLC Genomic Workbench v7.5.1, GeneiousR8 and Newbler v2.9. The earliest strain of EBOV 2014, H.sapiens-wt/ GIN/2014/Makona-Kissidougou-C15 (GenBank accession number KJ660346.2) was used as the reference genome. The synonymous substitutions, non-synonymous substitutions, and substitutions in non-coding regions were marked with coloured dots. 15.

16. 17.

Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). Bielejec, F. et al. SPREAD: spatial phylogenetic reconstruction of evolutionary dynamics. Bioinformatics 27, 2910–2912 (2011). Minin, V.N. et al. Smooth skyride through a rough skyline: Bayesian coalescentbased inference of population dynamics. Mol. Biol. Evol. 25, 1459–1471 (2008).

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100

KJ660347_Gueckedou-C07_2014-03-20_GIN-District-Town KJ660348_Gueckedou-C05_2014-03-19_GIN-District-Town KJ660346_Kissidougou-C15_2014-03-17_GIN-District-Town KM034550_EM095_2014-05-25_SLE-Kailahun-KissiTeng KM034555_G3676_2_2014-06-06_SLE-Kailahun-KissiTeng KM034559_G3680_1_2014-05-28_SLE-Kailahun-KissiTeng KM034561_G3683_1_2014-05-28_SLE-Kailahun-KissiTeng KM034562_G3686_1_2014-05-28_SLE-Kailahun-KissiTongi KM034563_G3687_1_2014-05-28_SLE-Kailahun-KissiTongi KM034552_EM098_2014-05-26_SLE-Kailahun-KissiTeng KM034553_G3670_1_2014-05-27_SLE-Kailahun-KissiTeng KM034557_G3677_2_2014-05-27_SLE-Kailahun-KissiTeng KM034560_G3682_1_2014-05-28_SLE-Kailahun-KissiTeng KM233043_EM120_2014-06-03_SLE-Kailahun-Jawie KM233044_EM121_2014-06-04_SLE-Kailahun-KissiKama KM233054_G3729_2014-06-07_SLE-Kailahun-Jawie KM233055_G3734_1_2014-06-07_SLE-Kailahun-Luawa KM233062_G3758_2014-06-11_SLE-Kailahun-Jawie KM233065_G3769_1_2014-06-12_SLE-Kailahun-Jawie KM233072_G3782_2014-06-14_SLE-Kailahun-KissiTeng KM233073_G3786_2014-06-14_SLE-Kailahun-KissiTeng KM233075_G3788_2014-06-14_SLE-Kailahun-KissiTeng KM233077_G3795_2014-06-15_SLE-Kailahun-Jawie KM233078_G3796_2014-06-15_SLE-Kailahun-Jawie KM233080_G3799_2014-06-15_SLE-Kailahun-Jawie KM233081_G3800_2014-06-15_SLE-Kailahun-Jawie KM233084_G3807_2014-06-15_SLE-Kailahun-Jawie KM233085_G3808_2014-06-15_SLE-Kailahun-Jawie KM233088_G3810_2_2014-06-17_SLE-Kailahun-Jawie KM233094_G3820_2014-06-15_SLE-Kailahun-Jawie KM233097_G3823_2014-06-15_SLE-Kailahun-Jawie KM233105_G3838_2014-06-17_SLE-Kailahun-Jawie KM233107_G3841_2014-06-17_SLE-Bo-Kakua KM034551_EM096_2014-05-26_SLE-Kailahun-KissiTeng KM034558_G3679_1_2014-05-28_SLE-Kailahun-KissiTeng KM233074_G3787_2014-06-14_SLE-Kailahun-KissiTeng KM233103_G3831_2014-06-16_SLE-Kailahun-KissiTeng J0156_T_2014-11-07_SierraLeone-WesternUrban-Freetown KM233035_EM104_2014-06-02_SLE-Kailahun-Jawie KM233036_EM106_2014-06-02_SLE-Kailahun-Jawie KM233037_EM110_2014-06-03_SLE-Kailahun-Jawie KM233038_EM111_2014-06-03_SLE-Kailahun-Jawie KM233039_EM112_2014-06-03_SLE-Kailahun-Jawie KM233040_EM113_2014-06-03_SLE-Kailahun-Jawie KM233041_EM115_2014-06-03_SLE-Kailahun-Jawie KM233047_EM124_3_2014-06-08_SLE-Kailahun-Jawie KM233049_G3707_2014-06-06_SLE-Kailahun-Jawie KM233050_G3713_2_2014-06-09_SLE-Kailahun-Njaluahun KM233053_G3724_2014-06-05_SLE-Kailahun-Jawie KM233056_G3735_1_2014-06-07_SLE-Kailahun-Jawie KM233058_G3750_1_2014-06-10_SLE-Kailahun-Jawie KM233063_G3764_2014-06-12_SLE-District-Town KM233064_G3765_2_2014-06-14_SLE-Kailahun-Jawie KM233071_G3771_2014-06-12_SLE-Kailahun-Mandu KM233076_G3789_1_2014-06-14_SLE-Kailahun-Jawie KM233079_G3798_2014-06-15_SLE-Kailahun-Jawie KM233082_G3805_1_2014-06-15_SLE-Kailahun-Jawie KM233089_G3814_2014-06-15_SLE-Kailahun-Jawie KM233091_G3817_2014-06-15_SLE-Kailahun-Jawie KM233092_G3818_2014-06-15_SLE-Kailahun-Jawie KM233093_G3819_2014-06-15_SLE-Kailahun-Jawie KM233106_G3840_2014-06-17_SLE-Kailahun-Jawie KM233108_G3845_2014-06-18_SLE-Kailahun-Jawie KM233109_G3846_2014-06-18_SLE-Kailahun-Jawie KM233111_G3850_2014-06-18_SLE-Kailahun-Jawie KM233113_G3856_1_2014-06-18_SLE-Kenema-Nongowa KM233116_NM042_1_2014-06-04_SLE-Kambia-Mambolo KM233042_EM119_2014-06-03_SLE-Kailahun-Jawie KM233070_G3770_2_2014-06-14_SLE-Kailahun-Jawie KM233100_G3826_2014-06-16_SLE-Kailahun-Jawie KM233101_G3827_2014-06-16_SLE-Kailahun-Jawie KM233086_G3809_2014-06-15_SLE-Kailahun-Jawie KM233090_G3816_2014-06-15_SLE-Kailahun-Jawie KM233095_G3821_2014-06-15_SLE-Kailahun-Jawie KM233096_G3822_2014-06-15_SLE-Kailahun-Jawie J0051_B_2014-10-06_SierraLeone-WesternUrban-Freetown J0053_T_2014-10-06_SierraLeone-WesternRural-Waterloo J0054_T_2014-10-06_SierraLeone-WesternRural-Waterloo J0092_B_2014-10-25_SierraLeone-WesternRural-Waterloo J0099_T_2014-10-28_SierraLeone-Kambia-Kambia J0137_T_2014-11-01_SierraLeone-WesternRural-Waterloo J0113_T_2014-10-30_SierraLeone-WesternUrban-Freetown J0007_B_2014-09-27_SierraLeone-WesternRural-Coldbath J0008_T_2014-09-29_SierraLeone-WesternUrban-Freetown J0016_T_2014-09-30_SierraLeone-WesternRural-Waterloo J0050_T_2014-10-05_SierraLeone-WesternRural-Coldbath J0066_T_2014-10-10_SierraLeone-WesternUrban-Adonkia J0089_T_2014-10-22_SierraLeone-WesternRural-Coldbath J0112_T_2014-10-31_SierraLeone-WesternUrban-Freetown J0134_T_2014-10-31_SierraLeone-WesternRural-Waterloo J0135_B_2014-11-02_SierraLeone-WesternRural-Waterloo J0024_T_2014-09-29_SierraLeone-WesternRural-Waterloo J0028_T_2014-10-02_SierraLeone-Bombali-Mapema J0001_B_2014-09-27_SierraLeone-WesternRural-Jui J0042_T_2014-10-04_SierraLeone-WesternRural-Waterloo J0005_B_2014-09-26_SierraLeone-WesternRural-Jui J0121_T_2014-10-29_SierraLeone-WesternUrban-Freetown J0173_B_2014-11-10_SierraLeone-WesternRural-Sulpon J0146_B_2014-11-06_SierraLeone-WesternUrban-Freetown J0147_B_2014-11-06_SierraLeone-WesternUrban-Freetown J0150_T_2014-11-06_SierraLeone-WesternUrban-Freetown J0157_T_2014-11-06_SierraLeone-WesternUrban-Freetown J0039_B_2014-10-03_SierraLeone-Portloko-MaforkiChiefdom J0154_B_2014-11-06_SierraLeone-WesternUrban-Freetown J0159_B_2014-11-05_SierraLeone-WesternRural-Waterloo J0149_T_2014-11-06_SierraLeone-WesternUrban-Freetown J0002_B_2014-09-27_SierraLeone-WesternUrban-Freetown J0041_T_2014-10-04_SierraLeone-WesternUrban-Freetown J0143_B_2014-11-03_SierraLeone-Kambia-Kambia J0144_B_2014-11-03_SierraLeone-Kambia-Kambia J0171_T_2014-11-10_SierraLeone-WesternRural-Allen J0174_T_2014-11-10_SierraLeone-WesternRural-Waterloo J0021_T_2014-09-29_SierraLeone-Bombali-Kambia KM233061_G3752_2014-06-10_SLE-Kailahun-KpejeBongre KM233099_G3825_2_2014-06-17_SLE-Kailahun-Malema KM233102_G3829_2014-06-16_SLE-Kailahun-Luawa KM233104_G3834_2014-06-17_SLE-Kailahun-KpejeWest KM233110_G3848_2014-06-18_SLE-Kailahun-Jawie KM233112_G3851_2014-06-18_SLE-Kenema-Nongowa KM233115_G3857_2014-06-18_SLE-Kailahun-Luawa J0123_T_2014-11-01_SierraLeone-WesternRural-Waterloo J0152_B_2014-11-06_SierraLeone-WesternRural-Waterloo J0160_B_2014-11-06_SierraLeone-WesternRural-Waterloo J0015_T_2014-09-26_SierraLeone-Bombali-Kambia J0017_T_2014-09-26_SierraLeone-Bombali-Kambia J0019_T_2014-09-25_SierraLeone-Bombali-Kambia J0020_T_2014-09-25_SierraLeone-Bombali-Kambia J0022_T_2014-09-25_SierraLeone-Bombali-Kambia J0096_B_2014-10-27_SierraLeone-WesternUrban-Freetown J0018_B_2014-09-30_SierraLeone-WesternRural-Hastings J0061_B_2014-10-09_SierraLeone-PortLoko-MarampaChiefdom J0073_T_2014-10-11_SierraLeone-PortLoko-MarampaChiefdom J0142_T_2014-11-03_SierraLeone-WesternUrban-Freetown J0114_B_2014-10-29_SierraLeone-PortLoko-MarampaChiefdom J0115_B_2014-10-29_SierraLeone-PortLoko-MarampaChiefdom J0029_T_2014-09-29_SierraLeone-PortLoko-MaforkiChiefdom J0164_B_2014-11-09_SierraLeone-WesternRural-Coldbath J0011_B_2014-09-29_SierraLeone-Portloko-Mayonkoli J0030_B_2014-10-03_SierraLeone-Portloko-MaforkiChiefdom J0140_B_2014-10-30_SierraLeone-WesternRural-Waterloo J0012_B_2014-09-29_SierraLeone-Portloko-MaforkiChiefdom J0049_T_2014-10-06_SierraLeone-PortLoko-MaforkiChiefdom J0163_T_2014-11-08_SierraLeone-PortLoko-KoyaChiefdom J0013_B_2014-09-28_SierraLeone-PortLoko-MarampaChiefdom J0014_T_2014-09-29_SierraLeone-Portloko-Malap J0048_T_2014-10-05_SierraLeone-WesternRural-Waterloo J0088_B_2014-10-20_SierraLeone-PortLoko-KoyaChiefdom J0118_B_2014-10-31_SierraLeone-WesternRural-Waterloo J0079_T_2014-10-16_SierraLeone-WesternRural-Coldbath J0165_T_2014-11-08_SierraLeone-WesternRural-Coldbath J0085_B_2014-10-17_SierraLeone-PortLoko-MarampaChiefdom J0101_B_2014-10-29_SierraLeone-PortLoko-MasimeraChiefdom J0003_B_2014-09-28_SierraLeone-Portloko-MasimeraChiefdom J0032_T_2014-09-30_SierraLeone-PortLoko-MarampaChiefdom J0074_T_2014-10-13_SierraLeone-WesternRural-Waterloo J0141_T_2014-11-03_SierraLeone-WesternRural-Johntop J0168_T_2014-11-10_SierraLeone-WesternRural-Waterloo J0038_B_2014-09-30_SierraLeone-PortLoko-MaforkiChiefdom J0102_B_2014-10-27_SierraLeone-PortLoko-Kambia J0103_B_2014-10-29_SierraLeone-PortLoko-Kambia J0104_T_2014-10-29_SierraLeone-PortLoko-MasimeraChiefdom J0105_T_2014-10-29_SierraLeone-PortLoko-MaforkiChiefdom J0044_T_2014-10-05_SierraLeone-WesternRural-Waterloo J0047_B_2014-10-05_SierraLeone-WesternUrban-Freetown J0124_T_2014-11-01_SierraLeone-WesternUrban-Freetown J0132_B_2014-10-31_SierraLeone-WesternUrban-Freetown J0087_B_2014-10-22_SierraLeone-PortLoko-MasimeraChiefdom J0078_T_2014-10-17_SierraLeone-WesternUrban-Freetown J0086_B_2014-10-22_SierraLeone-WesternRural-Jui J0043_T_2014-10-04_SierraLeone-WesternUrban-Freetown J0080_T_2014-10-17_SierraLeone-PortLoko-MaforkiChiefdom J0093_T_2014-10-24_SierraLeone-WesternRural-Johntop J0136_T_2014-11-01_SierraLeone-WesternRural-Rokel J0167_B_2014-11-08_SierraLeone-WesternUrban-Freetown J0059_T_2014-10-07_SierraLeone-WesternRural-KentJunction J0060_T_2014-10-06_SierraLeone-WesternUrban-Freetown J0065_T_2014-10-09_SierraLeone-WesternUrban-Freetown J0052_T_2014-10-07_SierraLeone-WesternRural-Rokel J0075_T_2014-10-17_SierraLeone-WesternRural-Waterloo J0077_T_2014-10-17_SierraLeone-WesternRural-Waterloo J0090_B_2014-10-22_SierraLeone-WesternUrban-Freetown J0094_T_2014-10-27_SierraLeone-WesternRural-Waterloo J0129_B_2014-11-01_SierraLeone-WesternUrban-Freetown J0151_B_2014-11-06_SierraLeone-WesternRural-Coldbath J0169_T_2014-11-09_SierraLeone-WesternUrban-Freetown J0153_B_2014-11-06_SierraLeone-WesternRural-Waterloo J0172_B_2014-11-10_SierraLeone-WesternRural-Hastings J0081_T_2014-10-16_SierraLeone-WesternRural-Waterloo J0108_T_2014-10-28_SierraLeone-WesternRural-Waterloo J0004_T_2014-09-28_SierraLeone-WesternRural-Cole J0006_B_2014-09-28_SierraLeone-WesternRural-Cole J0040_B_2014-10-04_SierraLeone-WesternUrban-Freetown J0100_B_2014-10-26_SierraLeone-Kambia-Kambia J0117_T_2014-10-31_SierraLeone-WesternRural-Waterloo J0133_T_2014-10-31_SierraLeone-WesternRural-Waterloo J0069_T_2014-10-09_SierraLeone-WesternRural-Waterloo J0170_B_2014-11-10_SierraLeone-WesternUrban-Freetown J0091_T_2014-10-25_SierraLeone-WesternUrban-Freetown J0148_B_2014-11-06_SierraLeone-WesternRural-Waterloo J0026_T_2014-10-02_SierraLeone-Bombali-Kamakoni J0098_T_2014-10-28_SierraLeone-WesternRural-Cole J0106_T_2014-10-28_SierraLeone-WesternRural-Waterloo J0116_T_2014-10-30_SierraLeone-WesternRural-KentJunction J0045_T_2014-10-05_SierraLeone-WesternUrban-Freetown J0064_T_2014-10-09_SierraLeone-WesternUrban-Freetown J0068_T_2014-10-09_SierraLeone-WesternRural-CalabaTown J0107_T_2014-10-29_SierraLeone-WesternUrban-Freetown J0009_T_2014-09-28_SierraLeone-PortLoko-Rosint J0010_B_2014-09-28_SierraLeone-PortLoko-MaforkiChiefdom J0023_B_2014-10-01_SierraLeone-WesternRural-Waterloo J0027_T_2014-10-02_SierraLeone-Bombali-Makumre J0031_B_2014-10-03_SierraLeone-Portloko-MaforkiChiefdom J0055_T_2014-10-06_SierraLeone-PortLoko-MaforkiChiefdom J0062_T_2014-10-09_SierraLeone-PortLoko-Romeni J0063_B_2014-10-09_SierraLeone-PortLoko-MaforkiChiefdom J0109_B_2014-10-31_SierraLeone-WesternUrban-Freetown J0162_B_2014-11-09_SierraLeone-Kambia-Kambia J0034_B_2014-09-30_SierraLeone-PortLoko-KoyaChiefdom J0067_T_2014-10-09_SierraLeone-PortLoko-MaforkiChiefdom J0130_T_2014-11-01_SierraLeone-PortLoko-MasimeraChiefdom J0131_T_2014-10-30_SierraLeone-PortLoko-MaforkiChiefdom J0166_T_2014-11-08_SierraLeone-WesternUrban-Freetown J0175_T_2014-11-10_SierraLeone-WesternRural-Waterloo J0037_B_2014-10-03_SierraLeone-Portloko-Rosint J0046_T_2014-10-06_SierraLeone-WesternRural-Waterloo J0076_T_2014-10-17_SierraLeone-WesternRural-Waterloo J0127_T_2014-10-30_SierraLeone-WesternUrban-Freetown J0128_T_2014-10-31_SierraLeone-WesternRural-Waterloo J0056_B_2014-10-07_SierraLeone-PortLoko-KoyaChiefdom J0057_B_2014-10-05_SierraLeone-PortLoko-MaforkiChiefdom J0139_T_2014-10-30_SierraLeone-WesternUrban-Freetown J0161_T_2014-11-07_SierraLeone-WesternUrban-Freetown J0025_T_2014-10-02_SierraLeone-WesternUrban-Freetown J0082_T_2014-10-16_SierraLeone-WesternRural-York J0122_T_2014-10-29_SierraLeone-WesternUrban-Freetown J0125_T_2014-10-31_SierraLeone-WesternRural-Rogbangba J0126_T_2014-10-31_SierraLeone-WesternRural-Rogbangba J0138_T_2014-11-01_SierraLeone-WesternUrban-Freetown J0145_T_2014-11-05_SierraLeone-WesternRural-Cole J0095_B_2014-10-27_SierraLeone-WesternUrban-Freetown J0155_T_2014-11-06_SierraLeone-WesternRural-Hastings J0110_T_2014-10-31_SierraLeone-WesternUrban-Freetown J0119_T_2014-10-30_SierraLeone-WesternUrban-Freetown J0097_T_2014-10-27_SierraLeone-WesternRural-Waterloo J0111_B_2014-10-30_SierraLeone-WesternUrban-Freetown J0120_B_2014-10-30_SierraLeone-WesternUrban-Freetown J0158_T_2014-11-07_SierraLeone-WesternRural-Grafton J0033_T_2014-10-03_SierraLeone-Portloko-MaforkiChiefdom J0036_T_2014-10-03_SierraLeone-Portloko-MaforkiChiefdom J0070_T_2014-10-09_SierraLeone-PortLoko-BuyaRomendeChiefdom J0072_T_2014-10-09_SierraLeone-PortLoko-BuyaRomendeChiefdom J0058_T_2014-10-07_SierraLeone-PortLoko-MaforkiChiefdom J0071_T_2014-10-09_SierraLeone-PortLoko-BuyaRomendeChiefdom J0035_B_2014-10-01_SierraLeone-PortLoko-BuyaRomendeChiefdom J0083_T_2014-10-17_SierraLeone-PortLoko-Masoyila J0084_T_2014-10-16_SierraLeone-PortLoko-KoyaChiefdom

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3.2.2

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3.2.5

8.0E-5 Extended Data Figure 1 | Phylogenetic tree of the 2014 EBOV inferred using MrBayes. The seven novel sublineages are highlighted using different colours. Previously described EBOV sequences are shown in grey. Posterior probability for each lineage is shown.

G2015

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LETTER RESEARCH 94

KJ660347|Gueckedou-C07|2014-03-20|GIN-District-Town KJ660348|Gueckedou-C05|2014-03-19|GIN-District-Town KJ660346|Kissidougou-C15|2014-03-17|GIN-District-Town KM034555|G3676.2|2014-06-06|SLE-Kailahun-KissiTeng KM034563|G3687.1|2014-05-28|SLE-Kailahun-KissiTongi KM034550|EM095|2014-05-25|SLE-Kailahun-KissiTeng KM034562|G3686.1|2014-05-28|SLE-Kailahun-KissiTongi KM034559|G3680.1|2014-05-28|SLE-Kailahun-KissiTeng KM034561|G3683.1|2014-05-28|SLE-Kailahun-KissiTeng KM233055|G3734.1|2014-06-07|SLE-Kailahun-Luawa KM233043|EM120|2014-06-03|SLE-Kailahun-Jawie KM233105|G3838|2014-06-17|SLE-Kailahun-Jawie KM233080|G3799|2014-06-15|SLE-Kailahun-Jawie KM233044|EM121|2014-06-04|SLE-Kailahun-KissiKama KM233074|G3787|2014-06-14|SLE-Kailahun-KissiTeng KM233103|G3831|2014-06-16|SLE-Kailahun-KissiTeng KM233075|G3788|2014-06-14|SLE-Kailahun-KissiTeng KM233088|G3810.2|2014-06-17|SLE-Kailahun-Jawie KM034558|G3679.1|2014-05-28|SLE-Kailahun-KissiTeng KM034551|EM096|2014-05-26|SLE-Kailahun-KissiTeng KM034557|G3677.2|2014-05-27|SLE-Kailahun-KissiTeng KM233062|G3758|2014-06-11|SLE-Kailahun-Jawie KM233107|G3841|2014-06-17|SLE-Bo-Kakua KM233078|G3796|2014-06-15|SLE-Kailahun-Jawie KM034552|EM098|2014-05-26|SLE-Kailahun-KissiTeng KM233073|G3786|2014-06-14|SLE-Kailahun-KissiTeng KM233094|G3820|2014-06-15|SLE-Kailahun-Jawie KM233085|G3808|2014-06-15|SLE-Kailahun-Jawie KM233097|G3823|2014-06-15|SLE-Kailahun-Jawie KM233081|G3800|2014-06-15|SLE-Kailahun-Jawie KM034553|G3670.1|2014-05-27|SLE-Kailahun-KissiTeng KM233072|G3782|2014-06-14|SLE-Kailahun-KissiTeng KM233084|G3807|2014-06-15|SLE-Kailahun-Jawie KM233077|G3795|2014-06-15|SLE-Kailahun-Jawie KM233065|G3769.1|2014-06-12|SLE-Kailahun-Jawie KM233054|G3729|2014-06-07|SLE-Kailahun-Jawie KM034560|G3682.1|2014-05-28|SLE-Kailahun-KissiTeng KM233091|G3817|2014-06-15|SLE-Kailahun-Jawie KM233063|G3764|2014-06-12|SLE-District-Town KM233053|G3724|2014-06-05|SLE-Kailahun-Jawie KM233106|G3840|2014-06-17|SLE-Kailahun-Jawie KM233079|G3798|2014-06-15|SLE-Kailahun-Jawie J0051|B|2014-10-06|SierraLeone-WesternUrban-Freetown' J0054|T|2014-10-06|SierraLeone-WesternRural-Waterloo' J0099|T|2014-10-28|SierraLeone-Kambia-Kambia' J0092|B|2014-10-25|SierraLeone-WesternRural-Waterloo' J0053|T|2014-10-06|SierraLeone-WesternRural-Waterloo' J0137|T|2014-11-01|SierraLeone-WesternRural-Waterloo' KM233108|G3845|2014-06-18|SLE-Kailahun-Jawie KM233049|G3707|2014-06-06|SLE-Kailahun-Jawie KM233086|G3809|2014-06-15|SLE-Kailahun-Jawie KM233096|G3822|2014-06-15|SLE-Kailahun-Jawie KM233090|G3816|2014-06-15|SLE-Kailahun-Jawie KM233095|G3821|2014-06-15|SLE-Kailahun-Jawie KM233082|G3805.1|2014-06-15|SLE-Kailahun-Jawie J0156|T|2014-11-07|SierraLeone-WesternUrban-Freetown' KM233116|NM042.1|2014-06-04|SLE-Kambia-Mambolo KM233093|G3819|2014-06-15|SLE-Kailahun-Jawie KM233036|EM106|2014-06-02|SLE-Kailahun-Jawie KM233056|G3735.1|2014-06-07|SLE-Kailahun-Jawie KM233038|EM111|2014-06-03|SLE-Kailahun-Jawie KM233071|G3771|2014-06-12|SLE-Kailahun-Mandu KM233111|G3850|2014-06-18|SLE-Kailahun-Jawie KM233100|G3826|2014-06-16|SLE-Kailahun-Jawie KM233101|G3827|2014-06-16|SLE-Kailahun-Jawie KM233109|G3846|2014-06-18|SLE-Kailahun-Jawie KM233035|EM104|2014-06-02|SLE-Kailahun-Jawie KM233050|G3713.2|2014-06-09|SLE-Kailahun-Njaluahun KM233092|G3818|2014-06-15|SLE-Kailahun-Jawie KM233064|G3765.2|2014-06-14|SLE-Kailahun-Jawie KM233037|EM110|2014-06-03|SLE-Kailahun-Jawie KM233047|EM124.3|2014-06-08|SLE-Kailahun-Jawie KM233113|G3856.1|2014-06-18|SLE-Kenema-Nongowa KM233041|EM115|2014-06-03|SLE-Kailahun-Jawie KM233058|G3750.1|2014-06-10|SLE-Kailahun-Jawie KM233076|G3789.1|2014-06-14|SLE-Kailahun-Jawie KM233070|G3770.2|2014-06-14|SLE-Kailahun-Jawie KM233042|EM119|2014-06-03|SLE-Kailahun-Jawie KM233040|EM113|2014-06-03|SLE-Kailahun-Jawie KM233039|EM112|2014-06-03|SLE-Kailahun-Jawie KM233089|G3814|2014-06-15|SLE-Kailahun-Jawie J0113|T|2014-10-30|SierraLeone-WesternUrban-Freetown' J0149|T|2014-11-06|SierraLeone-WesternUrban-Freetown' J0041|T|2014-10-04|SierraLeone-WesternUrban-Freetown' J0002|B|2014-09-27|SierraLeone-WesternUrban-Freetown' J0174|T|2014-11-10|SierraLeone-WesternRural-Waterloo' J0171|T|2014-11-10|SierraLeone-WesternRural-Allen' J0144|B|2014-11-03|SierraLeone-Kambia-Kambia'

99

3.1.1

99

98

88

J0008|T|2014-09-29|SierraLeone-WesternUrban-Freetown' J0005|B|2014-09-26|SierraLeone-WesternRural-Jui' J0173|B|2014-11-10|SierraLeone-WesternRural-Sulpon' J0121|T|2014-10-29|SierraLeone-WesternUrban-Freetown' J0146|B|2014-11-06|SierraLeone-WesternUrban-Freetown' J0147|B|2014-11-06|SierraLeone-WesternUrban-Freetown' J0134|T|2014-10-31|SierraLeone-WesternRural-Waterloo' J0001|B|2014-09-27|SierraLeone-WesternRural-Jui' J0042|T|2014-10-04|SierraLeone-WesternRural-Waterloo'

J0143|B|2014-11-03|SierraLeone-Kambia-Kambia'

J0157|T|2014-11-06|SierraLeone-WesternUrban-Freetown' J0150|T|2014-11-06|SierraLeone-WesternUrban-Freetown' J0154|B|2014-11-06|SierraLeone-WesternUrban-Freetown' J0039|B|2014-10-03|SierraLeone-Portloko-MaforkiChiefdom' J0159|B|2014-11-05|SierraLeone-WesternRural-Waterloo' J0135|B|2014-11-02|SierraLeone-WesternRural-Waterloo' J0066|T|2014-10-10|SierraLeone-WesternUrban-Adonkia' J0016|T|2014-09-30|SierraLeone-WesternRural-Waterloo' J0112|T|2014-10-31|SierraLeone-WesternUrban-Freetown' J0007|B|2014-09-27|SierraLeone-WesternRural-Coldbath' J0050|T|2014-10-05|SierraLeone-WesternRural-Coldbath' J0089|T|2014-10-22|SierraLeone-WesternRural-Coldbath' J0024|T|2014-09-29|SierraLeone-WesternRural-Waterloo' J0028|T|2014-10-02|SierraLeone-Bombali-Mapema' KM233112|G3851|2014-06-18|SLE-Kenema-Nongowa KM233115|G3857|2014-06-18|SLE-Kailahun-Luawa KM233061|G3752|2014-06-10|SLE-Kailahun-KpejeBongre KM233104|G3834|2014-06-17|SLE-Kailahun-KpejeWest KM233110|G3848|2014-06-18|SLE-Kailahun-Jawie KM233099|G3825.2|2014-06-17|SLE-Kailahun-Malema KM233102|G3829|2014-06-16|SLE-Kailahun-Luawa J0055|T|2014-10-06|SierraLeone-PortLoko-MaforkiChiefdom' J0175|T|2014-11-10|SierraLeone-WesternRural-Waterloo' J0166|T|2014-11-08|SierraLeone-WesternUrban-Freetown' J0063|B|2014-10-09|SierraLeone-PortLoko-MaforkiChiefdom' J0109|B|2014-10-31|SierraLeone-WesternUrban-Freetown' J0010|B|2014-09-28|SierraLeone-PortLoko-MaforkiChiefdom' J0009|T|2014-09-28|SierraLeone-PortLoko-Rosint' J0131|T|2014-10-30|SierraLeone-PortLoko-MaforkiChiefdom' J0130|T|2014-11-01|SierraLeone-PortLoko-MasimeraChiefdom' J0034|B|2014-09-30|SierraLeone-PortLoko-KoyaChiefdom' J0067|T|2014-10-09|SierraLeone-PortLoko-MaforkiChiefdom' J0027|T|2014-10-02|SierraLeone-Bombali-Makumre' J0155|T|2014-11-06|SierraLeone-WesternRural-Hastings' J0095|B|2014-10-27|SierraLeone-WesternUrban-Freetown' J0119|T|2014-10-30|SierraLeone-WesternUrban-Freetown' J0110|T|2014-10-31|SierraLeone-WesternUrban-Freetown' J0158|T|2014-11-07|SierraLeone-WesternRural-Grafton' J0097|T|2014-10-27|SierraLeone-WesternRural-Waterloo' J0120|B|2014-10-30|SierraLeone-WesternUrban-Freetown' J0111|B|2014-10-30|SierraLeone-WesternUrban-Freetown' J0070|T|2014-10-09|SierraLeone-PortLoko-BuyaRomendeChiefdom' J0072|T|2014-10-09|SierraLeone-PortLoko-BuyaRomendeChiefdom' J0033|T|2014-10-03|SierraLeone-Portloko-MaforkiChiefdom' J0058|T|2014-10-07|SierraLeone-PortLoko-MaforkiChiefdom' J0071|T|2014-10-09|SierraLeone-PortLoko-BuyaRomendeChiefdom' J0036|T|2014-10-03|SierraLeone-Portloko-MaforkiChiefdom' J0084|T|2014-10-16|SierraLeone-PortLoko-KoyaChiefdom' J0035|B|2014-10-01|SierraLeone-PortLoko-BuyaRomendeChiefdom' J0083|T|2014-10-17|SierraLeone-PortLoko-Masoyila' J0031|B|2014-10-03|SierraLeone-Portloko-MaforkiChiefdom' J0062|T|2014-10-09|SierraLeone-PortLoko-Romeni' J0023|B|2014-10-01|SierraLeone-WesternRural-Waterloo' J0162|B|2014-11-09|SierraLeone-Kambia-Kambia' J0037|B|2014-10-03|SierraLeone-Portloko-Rosint' J0046|T|2014-10-06|SierraLeone-WesternRural-Waterloo' J0076|T|2014-10-17|SierraLeone-WesternRural-Waterloo' J0127|T|2014-10-30|SierraLeone-WesternUrban-Freetown' J0128|T|2014-10-31|SierraLeone-WesternRural-Waterloo' J0056|B|2014-10-07|SierraLeone-PortLoko-KoyaChiefdom' J0161|T|2014-11-07|SierraLeone-WesternUrban-Freetown' J0057|B|2014-10-05|SierraLeone-PortLoko-MaforkiChiefdom' J0139|T|2014-10-30|SierraLeone-WesternUrban-Freetown' J0025|T|2014-10-02|SierraLeone-WesternUrban-Freetown' J0145|T|2014-11-05|SierraLeone-WesternRural-Cole' J0138|T|2014-11-01|SierraLeone-WesternUrban-Freetown' J0125|T|2014-10-31|SierraLeone-WesternRural-Rogbangba' J0122|T|2014-10-29|SierraLeone-WesternUrban-Freetown' J0126|T|2014-10-31|SierraLeone-WesternRural-Rogbangba' J0082|T|2014-10-16|SierraLeone-WesternRural-York' J0073|T|2014-10-11|SierraLeone-PortLoko-MarampaChiefdom' J0142|T|2014-11-03|SierraLeone-WesternUrban-Freetown' J0018|B|2014-09-30|SierraLeone-WesternRural-Hastings' J0114|B|2014-10-29|SierraLeone-PortLoko-MarampaChiefdom' J0115|B|2014-10-29|SierraLeone-PortLoko-MarampaChiefdom' J0061|B|2014-10-09|SierraLeone-PortLoko-MarampaChiefdom' J0011|B|2014-09-29|SierraLeone-Portloko-Mayonkoli' J0030|B|2014-10-03|SierraLeone-Portloko-MaforkiChiefdom' J0029|T|2014-09-29|SierraLeone-PortLoko-MaforkiChiefdom' J0164|B|2014-11-09|SierraLeone-WesternRural-Coldbath' J0140|B|2014-10-30|SierraLeone-WesternRural-Waterloo' J0013|B|2014-09-28|SierraLeone-PortLoko-MarampaChiefdom' J0014|T|2014-09-29|SierraLeone-Portloko-Malap' J0163|T|2014-11-08|SierraLeone-PortLoko-KoyaChiefdom' J0049|T|2014-10-06|SierraLeone-PortLoko-MaforkiChiefdom' J0012|B|2014-09-29|SierraLeone-Portloko-MaforkiChiefdom' J0118|B|2014-10-31|SierraLeone-WesternRural-Waterloo' J0085|B|2014-10-17|SierraLeone-PortLoko-MarampaChiefdom' J0101|B|2014-10-29|SierraLeone-PortLoko-MasimeraChiefdom' J0048|T|2014-10-05|SierraLeone-WesternRural-Waterloo' J0038|B|2014-09-30|SierraLeone-PortLoko-MaforkiChiefdom' J0105|T|2014-10-29|SierraLeone-PortLoko-MaforkiChiefdom' J0103|B|2014-10-29|SierraLeone-PortLoko-Kambia' J0104|T|2014-10-29|SierraLeone-PortLoko-MasimeraChiefdom' J0102|B|2014-10-27|SierraLeone-PortLoko-Kambia' J0079|T|2014-10-16|SierraLeone-WesternRural-Coldbath' J0165|T|2014-11-08|SierraLeone-WesternRural-Coldbath' J0088|B|2014-10-20|SierraLeone-PortLoko-KoyaChiefdom' J0003|B|2014-09-28|SierraLeone-Portloko-MasimeraChiefdom' J0168|T|2014-11-10|SierraLeone-WesternRural-Waterloo' J0074|T|2014-10-13|SierraLeone-WesternRural-Waterloo' J0141|T|2014-11-03|SierraLeone-WesternRural-Johntop' J0032|T|2014-09-30|SierraLeone-PortLoko-MarampaChiefdom' J0021|T|2014-09-29|SierraLeone-Bombali-Kambia' J0123|T|2014-11-01|SierraLeone-WesternRural-Waterloo' J0160|B|2014-11-06|SierraLeone-WesternRural-Waterloo' J0152|B|2014-11-06|SierraLeone-WesternRural-Waterloo' J0015|T|2014-09-26|SierraLeone-Bombali-Kambia' J0019|T|2014-09-25|SierraLeone-Bombali-Kambia' J0017|T|2014-09-26|SierraLeone-Bombali-Kambia' J0022|T|2014-09-25|SierraLeone-Bombali-Kambia' J0096|B|2014-10-27|SierraLeone-WesternUrban-Freetown' J0020|T|2014-09-25|SierraLeone-Bombali-Kambia' J0124|T|2014-11-01|SierraLeone-WesternUrban-Freetown' J0132|B|2014-10-31|SierraLeone-WesternUrban-Freetown' J0044|T|2014-10-05|SierraLeone-WesternRural-Waterloo' J0093|T|2014-10-24|SierraLeone-WesternRural-Johntop' J0043|T|2014-10-04|SierraLeone-WesternUrban-Freetown' J0136|T|2014-11-01|SierraLeone-WesternRural-Rokel' J0080|T|2014-10-17|SierraLeone-PortLoko-MaforkiChiefdom' J0090|B|2014-10-22|SierraLeone-WesternUrban-Freetown' J0052|T|2014-10-07|SierraLeone-WesternRural-Rokel' J0169|T|2014-11-09|SierraLeone-WesternUrban-Freetown' J0075|T|2014-10-17|SierraLeone-WesternRural-Waterloo' J0129|B|2014-11-01|SierraLeone-WesternUrban-Freetown' J0077|T|2014-10-17|SierraLeone-WesternRural-Waterloo' J0094|T|2014-10-27|SierraLeone-WesternRural-Waterloo' J0151|B|2014-11-06|SierraLeone-WesternRural-Coldbath' J0153|B|2014-11-06|SierraLeone-WesternRural-Waterloo' J0172|B|2014-11-10|SierraLeone-WesternRural-Hastings' J0087|B|2014-10-22|SierraLeone-PortLoko-MasimeraChiefdom' J0078|T|2014-10-17|SierraLeone-WesternUrban-Freetown' J0086|B|2014-10-22|SierraLeone-WesternRural-Jui' J0167|B|2014-11-08|SierraLeone-WesternUrban-Freetown' J0065|T|2014-10-09|SierraLeone-WesternUrban-Freetown' J0059|T|2014-10-07|SierraLeone-WesternRural-KentJunction' J0060|T|2014-10-06|SierraLeone-WesternUrban-Freetown' J0047|B|2014-10-05|SierraLeone-WesternUrban-Freetown' J0133|T|2014-10-31|SierraLeone-WesternRural-Waterloo' J0117|T|2014-10-31|SierraLeone-WesternRural-Waterloo' J0026|T|2014-10-02|SierraLeone-Bombali-Kamakoni' J0106|T|2014-10-28|SierraLeone-WesternRural-Waterloo' J0116|T|2014-10-30|SierraLeone-WesternRural-KentJunction' J0098|T|2014-10-28|SierraLeone-WesternRural-Cole' J0006|B|2014-09-28|SierraLeone-WesternRural-Cole' J0091|T|2014-10-25|SierraLeone-WesternUrban-Freetown' J0148|B|2014-11-06|SierraLeone-WesternRural-Waterloo' J0170|B|2014-11-10|SierraLeone-WesternUrban-Freetown' J0069|T|2014-10-09|SierraLeone-WesternRural-Waterloo' J0004|T|2014-09-28|SierraLeone-WesternRural-Cole' J0081|T|2014-10-16|SierraLeone-WesternRural-Waterloo' J0108|T|2014-10-28|SierraLeone-WesternRural-Waterloo' J0100|B|2014-10-26|SierraLeone-Kambia-Kambia' J0040|B|2014-10-04|SierraLeone-WesternUrban-Freetown' J0045|T|2014-10-05|SierraLeone-WesternUrban-Freetown' J0068|T|2014-10-09|SierraLeone-WesternRural-CalabaTown' J0064|T|2014-10-09|SierraLeone-WesternUrban-Freetown' J0107|T|2014-10-29|SierraLeone-WesternUrban-Freetown'

3.1.2

76

3.2.5

72

3.2.2

97

3.2.3

58

3.2.1

68

8.0E-5 Extended Data Figure 2 | Maximum likelihood tree of 2014 EBOV constructed using RAxML.

G2015

Macmillan Publishers Limited. All rights reserved

3.2.4

RESEARCH LETTER KJ660347|guinea|20140320 KJ660348|guinea|20140319 KJ660346|guinea|20140317 1 0.9868 0

1

1

KM034555|sierraleone_kailahun|20140606 KM034554|sierraleone_kailahun|20140527 KM034550|sierraleone_kailahun|20140525 KM034558|sierraleone_kailahun|20140528 KM034552|sierraleone_kailahun|20140526 KM233088|sierraleone_kailahun|20140617 KM233068|sierraleone_kailahun|20140616 KM233044|sierraleone_kailahun|20140604 KM233054|sierraleone_kailahun|20140607 KM233037|sierraleone_kailahun|20140603 KM233059|sierraleone_kailahun|20140612 KM233058|sierraleone_kailahun|20140610 KM233047|sierraleone_kailahun|20140608 KM233083|sierraleone_kailahun|20140620 KM233060|sierraleone_kailahun|20140614 KM233051|sierraleone_kailahun|20140611 KM233052|sierraleone_kailahun|20140613 KM233082|sierraleone_kailahun|20140615 0.7374 0.9999

0.125 0.8576 0 8576

1 1

1 0.3253 0 3253

0.998

westernurban_freetown|20141107 westernurban_freetown|20141007 kambia_kambia|20141028 westernrural_waterloo|20141101 westernrural_waterloo|20141006 westernrural_waterloo|20141006 westernrural_waterloo|20141025 westernrural_waterloo|20140929 bombali_mapema|20141002 westernurban_freetown|20141107 kambia_kambia|20141104 kambia_kambia|20141104 westernrural_allen|20141110 westernrural_waterloo|20141111 westernurban_freetown|20141004 westernurban_freetown|20140927 westernrural_waterloo|20141103 westernurban_freetown|20141031 westernrural_waterloo|20140930 westernrural_coldbath|20141023 westernrural_coldbath|20141005 westernrural_coldbath|20140927 westernurban_adonkia|20141010 westernrural_jui|20140927 westernrural_waterloo|20141004 westernurban_freetown|20141106 westernurban_freetown|20141106 portloko_maforkichiefdom|20141003 westernurban_freetown|20141107 westernrural_waterloo|20141105 westernrural_waterloo|20141031 westernurban_freetown|20140929 westernurban_freetown|20141030 westernrural_jui|20140926 westernurban_freetown|20141107 westernurban_freetown|20141107 westernurban_freetown|20141029 westernrural_sulpon|20141111

3.1.1

3.1.2

KM233036|sierraleone_kailahun|20140602 KM233053|sierraleone_kailahun|20140605 KM233048|sierraleone_kailahun|20140609 KM233115|sierraleone_kailahun|20140618

0.0158 0.015 0 0.0 0 5 58 8 0.9951 0 9951

0.9418 0.9 0 .9 94 418

0.2772

1

0.6851 0 685 0.685 51 51

1

0.1715 0 1715

0.2061 0.206 0 206 2061 61 6 1

0.9999 0 9999

0 9241 0.9241

1

Locations

Waterloo 0 1248 0.1248

Freetown Maforki chiefdom

0 081 0.081

1

Rest of Western Rest of Port loko 0.9922 0 9922 2 0 9874 0.9874

Bombali Kambia

0.2108

1 0 997 0.997 74 7 4 0.9974

0.8128 0 8128

0.07 1

Mar

Apr

May

Jun

Jul

Aug

Sep

westernrural_hastings|20140930 portloko_marampachiefdom|20141010 westernurban_freetown|20141103 portloko_marampachiefdom|20141012 portloko_marampachiefdom|20141029 portloko_marampachiefdom|20141029 portloko_maforkichiefdom|20140929 westernrural_coldbath|20141109 portloko_maforkichiefdom|20141003 portloko_mayonkoli|20140929 westernrural_waterloo|20141030 portloko_malap|20140929 portloko_marampachiefdom|20140928 portloko_maforkichiefdom|20140929 portloko_koyachiefdom|20141108 portloko_maforkichiefdom|20141007 portloko_maforkichiefdom|20140930 portloko_kambia|20141027 portloko_kambia|20141029 portloko_maforkichiefdom|20141029 portloko_masimerachiefdom|20141029 westernrural_waterloo|20141031 westernrural_waterloo|20141005 westernrural_coldbath|20141108 westernrural_coldbath|20141016 portloko_koyachiefdom|20141020 portloko_masimerachiefdom|20141029 portloko_marampachiefdom|20141017 portloko_masimerachiefdom|20140928 portloko_marampachiefdom|20140930 westernrural_waterloo|20141013 westernrural_waterloo|20141111 westernrural_johntop|20141104 westernrural_waterloo|20141001 kambia_kambia|20141109 portloko_rosint|20141003 westernrural_waterloo|20141007 westernrural_waterloo|20141017 westernrural_waterloo|20141031 westernurban_freetown|20141030 portloko_koyachiefdom|20141008 westernurban_freetown|20141030 portloko_maforkichiefdom|20141005 westernurban_freetown|20141108 westernurban_freetown|20141002 westernrural_york|20141016 westernrural_rogbangba|20141031 westernrural_rogbangba|20141031 westernrural_cole|20141106 westernurban_freetown|20141029 westernurban_freetown|20141101 westernurban_freetown|20141108 westernrural_waterloo|20141111 bombali_makumre|20141002 westernurban_freetown|20141031 portloko_maforkichiefdom|20141006 portloko_koyachiefdom|20140930 portloko_maforkichiefdom|20141009 portloko_rosint|20140928 portloko_romeni|20141010 portloko_maforkichiefdom|20141003 portloko_maforkichiefdom|20140928 portloko_maforkichiefdom|20141010 portloko_masimerachiefdom|20141102 portloko_maforkichiefdom|20141030 westernrural_grafton|20141108 westernrural_waterloo|20141028 westernurban_freetown|20141030 westernurban_freetown|20141030 westernrural_hastings|20141107 westernurban_freetown|20141027 westernurban_freetown|20141031 westernurban_freetown|20141030 portloko_maforkichiefdom|20141003 portloko_maforkichiefdom|20141003 portloko_buyaromendechiefdom|20141009 portloko_maforkichiefdom|20141008 portloko_buyaromendechiefdom|20141009 portloko_buyaromendechiefdom|20141009 portloko_buyaromendechiefdom|20141001 |portloko_koyachiefdom|20141016 portloko_masoyila|20141018 westernrural_waterloo|20141107 westernrural_waterloo|20141107 westernrural_waterloo|20141101 westernurban_freetown|20141027 bombali_kambia|20140929 bombali_kambia|20140926 bombali_kambia|20140926 bombali_kambia|20140925 bombali_kambia|20140925 bombali_kambia|20140925 westernrural_johntop|20141024 1 portloko_maforkichiefdom|20141018 westernrural_rokel|20141101 westernurban_freetown|20141004 westernrural_waterloo|20141016 westernrural_waterloo|20141028 westernurban_freetown|20141005 westernurban_freetown|20141029 westernrural_calabatown|20141009 westernurban_freetown|20141009 bombali_kamakoni|20141002 westernrural_waterloo|20141028 westernrural_kentjunction|20141030 westernrural_cole|20141028 westernrural_cole|20140928 westernrural_cole|20140928 kambia_kambia|20141026 westernrural_waterloo|20141031 westernrural_waterloo|20141106 westernurban_freetown|20141025 westernurban_freetown|20141004 westernrural_waterloo|20141031 westernrural_waterloo|20141009 westernurban_freetown|20141110 westernurban_freetown|20141102 westernurban_freetown|20141102 westernrural_waterloo|20141005 westernurban_freetown|20141005 westernurban_freetown|20141108 westernrural_kentjunction|20141008 westernurban_freetown|20141009 westernurban_freetown|20141006 portloko_masimerachiefdom|20141023 westernrural_jui|20141023 westernurban_freetown|20141018 westernurban_freetown|20141109 westernrural_rokel|20141008 westernrural_hastings|20141111 westernrural_waterloo|20141107 westernurban_freetown|20141023 westernurban_freetown|20141102 westernrural_waterloo|20141017 westernrural_coldbath|20141107 westernrural_waterloo|20141017 westernrural_waterloo|20141027

Oct

Nov

3.2.2

3.2.3

3.2.5

3.2.1

3.2.4

Dec

Extended Data Figure 3 | Phylogeographic inference of the 2014 EBOV using BEAST. Previously described EBOV sequences are shown in grey. Posterior probability for each lineage is shown.

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LETTER RESEARCH 1196 GG T C T A T T GA T T G T C A A A A C AG T A C T T GA T C A T A T C C T A C A A A AGA C AGA A CGAGGAG T T CG T C T C C A T C C T C T T GC A AGGA C CGC C A AGG T A

J0150, J0157

GG T C T A T T GA T T G T C A A A A C AG T A C T T GA T C A T A T C C T A C A A A AGA C AGA A CGAGGAG T T CGC C T C C A T C C C C T T GC A AGGA C CGC C A AGG T A

A A A A A T GAGG T GA A C T C C T T C A AGGC T GC A C T C AGC T C C C T GGC C A AGC A T GGAGAG T A T GC T C C T T T CGC C CGA C T T T T GA A C C T T T C T GG A A A A A T GAGG T GA A C T C C T T C A AGGC T GC A C T C AGC T C C C T GGC C A AGC A T GGAGAG T A T GC T C C C T T CGC C CGA C T C T T GA A C C T T T C T GG

1472 AG T A A A T A A T C T T GAGC A T GG T C T T T T C C C T C A A C T G T CGGC A A T T GC A C T CGGAG T CGC C A C AGC C C A CGGGAGC A C C C T CGC AGGAG T A A AG T A A A C A A T C T T GAGC A T GG T C T T T T C C C T C A A C T G T CGGC A A T T GC A C T CGGAG T CGC C A C AGC C C A CGGGAGC A C C C T CGC AGGAG T A A

5458 A A T T GC A A T A A T T GA C T C AGA T C C AG T T T T A C AGA A T C T T C T C AGGGA T AG T GA T A A C A T C T T A A T T GC A A T A A T T GA C T C AGA T C C AG T T T T A C AGA A T C T T C T C AGGGA T AG T GA C A A C A T C C T

J0169

T T T A A T A A T C CG T C T A C T AGA AGAGA T A C T T C T A A T T GA T C A A T A T A C T A A AGG T GC T T T A C A C C A T T G T C T C T T T T C T C T C C T A A A T G T AG T C C A A T A A T C CG T C T A C T AGA AGAGA T A C T T C C A A T T GA T C A A T A C A C T A A AGG T GC T T T A C A C C A C T G T C T C C C C T C T C T C C C A A A T G T AG

5704 AGC T T A A C A A A AGA C T C A T A A T A T A C C T G T T T T T A A A AGA T T GA T T GA T GA A AGA T C A T GA C T A A T A A C A T T A C A A A C A A T C C T A C T A T A A T AGC C T A A C A A A AGA C T C A C A A T A T A C C T G T T T T T A A A AGA T T GA T T GA T GA A AGA T C A T GA C T A A T A A C A T T A C A A A C A A T C C T A C T A T A A T

8004

J0127

8096

T T GGAG T T A C AGG T G T T A T A A T T GC AG T T A T CGC T T T A T T C T G T A T A T GC A A A T T T G T C T T T T AG T C T T T C T T C AGA T T G T T T C A CGGC A A A A T T GGAGC C A C AGG T GC T A T A A T T GC AG T T A T CGC T T T A T T C T G T A T A T GC A A A T T T G T C C T C T AG T C T T T C T T C AGA T CG T T T C A CGGC A A A A

9475

J0024, J0028

T C AG T A C T A T A A T C A C T C T C A T T T C A A A T T GA T A AGA T A T GC A T A A T T GC C T T A A T A T A T A A AGAGG T A T GA T A T A A C C C A A A C A T T GA C C A A A T C AG T A C T A T A A T C A C T C T C A T T T C A A A T CGA C A AGA T A T GC A C A A T T GC C T C A A T A T A T A A AGAGG T A T GA T A T A A C C C A A A C A T T GA C C A A A

9660 GA A A A T C A T A A T C T CG T A T CGC T CGC A A T A T A A C C T GC C A AGC A T A C C T C T T GC A C A A AG T GA T T C T T G T A C A C A A A T A A T G T T T GA C T C T A GA A A A T C A T A A T C T CG T A T CGC T CGC A A T A T A A C C T GC C A AGC A T A C C T C T T GC A C A A AG T GA C T C T T G T A C A C A A A C A A T GC C T GA C T C T A

Extended Data Figure 4 | Original sequencing results of the serial TwC substitutions using the Sanger method. All of the four regions including serial T . C substitutions were sequenced using the Sanger method with the primers provided in Extended Data Table 1.

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Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER

NS

S

All

619

1201

3.1.1

17

32

3.1.2

124

3.2.1

27

3.2.2

43

226

3.2.5 0

8

28 19

146 282

181

45 187

324

20%

7

86

134

143

3.2.4

65

14

84

3.2.3

605

57

60%

80%

8 3 4 26

226

40%

a

IG

UTR

9 100%

b Gene-specific dN/dS Gene

dN/dS

95% HPD

NP VP35 VP40 GP VP30 VP24 L

0.4069 0.4497 0.2339 0.6065 0.2359 0.1114 0.2463

(0.2429, 0.6324) (0.2409, 0.7548) (0.0839, 0.5029) (0.3828, 0.9047) (0.0938, 0.4782) (0.0064, 0.4907) (0.1777, 0.3308)

c Lineage-specific dN/dS Lineage

dN/dS

95% HPD

3.1.1 3.1.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5

0.5847 0.2769 0.4242 0.2087 0.3869 0.2879 0.3414

(0.2096, 1.2562) (0.1593, 0.4415) (0.1822, 0.8202) (0.1003, 0.3762) (0.2179, 0.6263) (0.1837, 0.4255) (0.2301, 0.4839)

Extended Data Figure 5 | Synonymous and non-synonymous substitutions of the 2014 EBOV. a, Distribution of synonymous and non-synonymous substitutions in different lineages. The numbers of substitutions are labelled within bars. NS, non-synonymous; S, synonymous; UTR, UTR region; IG,

G2015

intergenic. b, Gene-specific global dN/dS estimates. The dN/dS and 95% highest posterior density interval were calculated using HyPhy. c, Lineagespecific global dN/dS estimates.

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LETTER RESEARCH Extended Data Table 1 | Primers designed for the confirmation of the serial T w C substitutions

Sample ID

Target region

Primer

J0150 & J0157

1139-1474

J0169

5434-5731

J0127

7967-8200

J0024 & J0028 

9401-9783

GGACATGATGCCAACGATGC(+) ATTTACTCCTGCGAGGGTGC(-) GTCTTCCAGCTGTGGTTGAGA(+) AAGATTGACATTTGAATCACCGT(-) TGGTGGACAGGATGGAGACA(+) GGCTATGTTTGAAGCTCCAGTG(-) CCTTCTACTTGATCACAATACTCCG(+) CCTCCTCCACAACTTGAAGCA(-)

G2015

Macmillan Publishers Limited. All rights reserved