Cell Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone Graphical Abstract Authors Daniel J. Park, Gytis Dudas, Early outbreak Prolonged epider Chr Weth Nea fambaut So S~ > Robert F. Garry, Pardis C. Sabeti eS. eA ct Correspondence € dpark@broadinstitute.org (D.u.P.), arrambaut@ed.ac.uk (A.R.), ake pardis@broadinstitute.org (P.C.S.) & Ss Ebola virus genomes from 232 patients ‘sampled over 7 months in Sierra Leone * bet reeled rutations, Ppecoliperoeetebeanail were sequenced. Transmission of Ps intrahost genetic variants suggests a . Mutations in Ebola virus sufficiently high infectious dose during within each pation transmission. The human host may have 5 neutral mutations caused direct alterations to the Ebola = ae i mildly deleterious mutations ation sample * " high deleterious mutations Highlights # In Sierra Leone, transmission has primarily been within- country, not between-country Infectious doses are large enough for intrahost variants to transmit between hosts ‘* Aprolonged epidemic removes deleterious mutations from the viral population ‘© There is preliminary evidence for human RNA editing effects on the Ebola genome ® Park ot al,, 2015, Coll 161, 1516-1526 eter CellPressCell Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone Danie! J. Park,’-*'-" Gytis Dudas,” Shirlee Wobl,'-*" Augustine Goba,’-*! Shannon L.M. Whitmer," Kristian G. Andersen,” Rachel S. Seaifon,'"” Jason T. Ladner," Jeffrey R. Kugelman,” Christian B. Matranga,’ Sarah M. Winnicki,'" James Qu,’ Stephen K. Gire,"-) Adrianne Gladden-Young,” Simbirie Jalloh,” Dolo Nosamiofan,’ Nathan L. Yozwiak,'® Lina M, Moses,” Pan-Pan Jiang,’ Aaron E. Lin,’ Stephen F. Schaffner, '" Brian Bird,” Jonathan Towner,” Mambu Mamoh, Michael Gbakie,* Lansana Kanneh, David Kargbo, James LB. Massally,* Fatima K. Kamara,“ Edwin Konuwa,’ Josephine Sellu,* Abdul A. Jalloh,” Ibrahim Mustapha,’ Momoh Foday,* ‘Mohamed Villah,* Bobbie R. Erickson,” Tara Sealy,” Dianna Blau,” Christopher Paddock,” Aaron Brault,” Brian Amman,° Jane Basile,” Scott Bearden,” Jessica Belser,” Eric Bergeron,” Shelley Campbell,” Ayan Chakrabarti,” kimberly Dodd, ° ‘Mike Flint,” Aridth Gibbons,” Christin Goodman,” John Klena,® Laura McMullan,” Laura Morgan,” Brandy Russell,” Johanna Salzer," Angela Sanchez,” David Wang,” Irvin Jungreis,” Christopher Tomkins-Tinch," Andrey Kisiyuk,* Michael F. Lin,” Sinead Chapman,’ Bronwyn MacInnis, Ashley Matthews," James Bochicchio,' Lisa E. Hensiey,"" Jens H. Kuhn,” Chad Nusbaum,’ John S. Schieffelin,” Bruce W. Birren,” Marc Forget,'® Stuart T. Nichol, ‘Gustavo F. Palacios,’ Daouda Ndlaye,'° Christian Happi,'* Sahr M. Gevao,'® Mohamed A. Vandi,'® Brima Kargbo,"° Edward C. Holmes,” Trevor Bedford,'° Andreas Gnirke, Ute Stroher,°"" Andrew Rambaut,”'""°2" Robert F. Garry,9? and Pardis C. Sabeti!-=" "Broad Insitute of Harvard and MIT, 75 Ames Street, Cambridge, MA 02142, USA =jpsttute of Evolutionary Biology, Ashworth Laboratoies, University of Edinburgh, Edinburgh EH 3FL, UK Harvard University, 82 Oxford Stroet, Cambridge, MA 02138, USA *#Kenema Government Hospital, Kenema, Sierra Leone "National Center for Emerging and Zoonotic Infectious Diseases and National Cantar for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1800 Cifon Road NE, Mailstop-G'14, Atlanta, GA 30333, USA "Scripps Translational Science institute, The Scripps Research Institute, 3644 N Torrey Pines Cour, La Jolla, CA 82087, USA Massachusetts Insitute of Technology, 77 Massactusetts Avenue, Cambridge, MA 02139, USA US Army Medical Research Insitute of Infectious Diseases, 1425 Porter Stroct, Fort Detrick, Froderck, MD 21702, USA Tulane University, 1430 Tulane Avenue, SL-38, New Orleans, LA 70112, USA ‘pNAnexus, 1975 West El Camino Fea, Suite 101, Mountain View, CA'94040, USA ‘integrated Research Facity at Fort Detrick, Division of Cincal Research, National Institue of Allergy and Infectious Diseases, National Institutes of Health, B-8200 Research Plaza, Fort Detrick, Frederick, MD 21702, USA "Medecins Sans Frontieres, Rue de Arbre Bént 46, 1050 Bruxoes, Belgium “Université Cheikh Anta Diop, BP 5005, Dakar, Sénégal ‘Radeemers University Nigeria, KM 46 Lagos-Ibadan Expressway, Redemption City, Ogun State, Nigeria "University of Sierra Leone, A.J. Momnoh St, Tower Hil, Freetown, Sita Leone "Sierra Leone Ministry of Health and Sanitation, Youyi Building, Freetown, Sierra Leone "University of Sydney, Johns Hopkins Drive, Camperdown NSW 2050, Australia + Fred Hutchinson Cancer Research Center, 110 Fairview Avenue North, Seats, WA 98108, USA + Contre for Immunology, Infection and Evolution, University of Edinburgh, Ashworth Laboratories, Edinburgh EH9 3FL, UK ‘Fogarty Intemational Center, National institutes of Health, °1 Center Drive, MSC 2220 Bethesda, MD 20882, USA iGo author 2200-senior author “Correspondence: dpari@broacinsttuis.org (DLP), a ambautided ac uk (AR, parcistbroadinsttute.org (PCS) http:/cx. dl org/10.1016/ cel. 2015.06.007 ‘This isan open access arise under the CC BY-NO-ND license (nip//creaiwecommons.orglicenses/by-no-nd/4.0 ‘SUMMARY ‘The 2013-2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients ‘sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evi- 1816 Coll 161, 1516-1526, June 18, 2015 ©2015 The Authors dence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation ‘of nonsynonymous mutations over time. Finally, ‘we note changes in the mucin-like domain of EBOV ‘lycoprotein that merit further investigation. These findings clarify the movement of EBOV within the ro- 0 during prolonged human-to-human transmission. DownesINTRODUCTION ‘The 2013-2015 Wester African Ebola virus disease (EVD) epidemic, caused by the Ebola virus (EBOV) Makona variant (Kuhn et al, 2014) is the largest EVD outbreak to date, with 26,848 cases and 11,017 deaths documented as of May 8, 2015 (WHO, 2015). The outbreak, first declared in March 2014 in Guinea and traced back to the end of 2013 (Baize et al, 2014), has also devastated the neighboring countries of Sierra, Leone and Liberia, with additional cases scattered across the globe. Never before has an EBOV variant been transmitted ‘among humans for such a sustained period of time. Published EBOV Makona genomes from clinical samples ob- tained early in the outbreak i Guinea (theee patients) and Sierra, Leone (78 patients) (Baize ota, 2014; Gre ot al, 2014) demon- strated that near-real-time sequencing could provide valuable Information to researchers involved in the global outbreak response. Analysis of these genomes revealed that the outbreak likely originated from a single introduction into the human popu- lationin Guinea at the end of 2013 and was then sustained excli- sivoly by human-to-human transmissions. Genomic sequencing further allovied the identification of numerous mutations: ‘emerging in the EBOV Makona genome over time. As a conse- uence, the evolutionary rate of the Makona variant over the: time span ofthe early phase ofthe outbreak could be estimated land predictions made about the potential of this new EBOV variant to escape current candidate vaccines, therapeutics, and diagnostics (Kugelman etal, 20154). While the insights gleaned from sequencing early in the ‘outbreak informed public health efforts (Alizon et al, 2014; Stax ler et al, 2014; Volz anc Pond, 2074), the continued human- ‘to-human spread of the virus raises questions about ongoing evolution and transmission of EBOV. Our laboratory teams in Si- ferra Leone, at Kenema (Kenema Government Hospital [KGH) and at Bo (US Centers for Disease Control and Prevention ICDC), continued to perform active diagnosis and surveillance in Sierra Leone following our inital study (Sie eta, 2011). After ‘8 6-month delay of sample shipment due to regulatory uncer- tainty about inactivation protocols, we again began to determine EBOV genome sequences. We have sequenced samples at high depth and with technical replicates. to characterize genetic diversity of EBOV both within (ntrahost) and between (nternost) individuals. To support global outbreak termination efforts, we publicly released these genomes prior to publication as they wore generated, starting with a frst set of 45 sequences in December 2014 and continuing with regular releases of hun- dreds of sequences through May 2016. Here, we provide an analysis of 232 new, coding-complete EBOV Makona genomes from Sierra Leone. We compared these ‘genomes to 86 previously available genomes: 78 unique ge- homes from Sierra Leone (Gire et al, 2014), 3 genomes from Guinea (Beize et al, 2014), and 5 from healthcare workers in- fected in Siera Leone and treated in Europe. We use this com bined data set obtained from 318 EVD pationts during the height ofthe epidemic in Sierra Leone and Guinea to better understand EBOV transmission within Sierra Leone and between countries. In addition, we use it to understand vial population dynamics within individual hosts, the impact of natural selection, and the: Characteristics of the now hundreds of new mutations that have emerged over the longer course ofthe epidemic. RESULTS 232 New Ebola Virus Makona Genomes from Sierra Leone We performed massively parallel genome sequencing on 673 ‘samples from two EVD patient cohorts. The first cohort included 8575 blood samples from 484 EVD patients confirmed by labora- tory staff at KGH from June 16 through September 28, 2014. The ‘second cohort included blood samples from 88 EVD patients, from throughout Sierra Leone confirmed at Bo by CDC labora- tory staf from August 20, 2014 through January 10, 2018. Sam- ples from both EVD cohorts were sequenced using previously described methods (Experimental Procedures; Matranga et al. 2014; Gire ot al., 2014). We implemented a new computational pipeline, viral 'ngs:v1.0.0, for vial genomic de novo assembly, itrahost variant caling, and genome analysis and annotation. This pipeline is available via open-source software (Park eta, 2015) and utilizes ‘a generalized workflow engine to run on a wide variety of com- puter harcware configurations (Koster and Rahman, 2012) Through a partnership with DNAnexus, this pipeline is also avail- able in a secure cloud-compute environment to enable consis- tent analyses across laboratories with limited computational resources (Experimental Procedures), Using this pipeline, we successfully assembled 232 EBOV Ma- kona coding-complete genomes (150 from KGH and 82 from the CDC cohort, spanning June 16 to December 26, 2014). Each assembled sequence was at least 18.5 kb in length, with a ‘maximum of 6% ambiguous base calls per genome, The median assembly had 374% coverage, was 18.9 kb long, and had no ‘ambiguous bases. Despite extensive sequencing, successful full-genome assembly was difficult to obtain from the KGH ‘cohort (73% failed ganome assemblies; 374% mean coverage; Table S1), compared to a previous coho from the same labora- tory, described in Gir etal (2014) (1196 failed genome assern- bles; 2,000x mean coverage). The high assembly failure rate of the more recent KGH cohort is likely due to the mandatory in- ‘county implementation of anew EBOV sample deactivation pro- tocol and to long delays for sample shipments amidst the outbreak response (see Experimental Procedures). In contrast, only 796 of samples from the CDC cohort failed to assemble. However, these samples had been pre-selected for sequencing ‘based on high EBOV titers, as estimated by qPCR. In adltion, the CDC cohort samples were collected more recently, did not remain in ysis buffer for an extended period, and were subjected to. different sample deactivation protocol than the KGH cohort samples. While we are continuing attempts to glean genomic informa- tion from compromised samples of the recent KGH cohort, Important information may have been lost In particular, samples. from many EBOV-infected health-care workers at KGH, which ‘could provide important insights into hospital-based transmis- sions, were compromised. In combination with the 86 previously published EBOV Ma- kona genomes (Gire et al,, 2014), we analyzed a total of 318 (Col 161, 1518-1526, June 18, 2015 ©2015 The Authors. 1517 CellCell genomes (see Experimental Procedures), all aligned against the earliest sampled Guinean genome (GenBank: KJ660346.2). In this set, we observed 464 single-nucleotide polymorphisms (SNPs; 125 nonsynonymous, 176 synonymous, and 163 non- Coding). We also observed five single-base insertions and two \double-base insertions in noncoding regions. We mapped all Of the variants to primer-binding sites for known sequence- ‘based diagnostics (Kugelman etal, 2075a) and found no muta- tions in these sites that were present in more than one Siewa Leonean sample (Tabie $2), We constructed a second, independent genome library for {each of 150 high-quality samples from the KGH cohort to reliably determine intrahost single-nucleotide variants (SNVs) at low frequencies (Gire otal, 2014). We identified 247 iSNVs 25 inser- tion/delations that were excluded from all analyses, 73 nonsy- ‘nonymous, 71 synonymous, and 78 noncoding), including 21 ISNVs shared by mutipe patients. Very recently, anther 175 EBOV Makona genomes were published based on a cohort from Sierra Leone, mostly sampled from the area of Freetown inthe Fall of 2014 (Tong eta, 2015). ‘Although these data were not included in our analyses, they are Unlikely to significantly ater our primary findings (Figure S') 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 (Cire ot al., 2014) from late May to early June, 2014. The first reported EVD cases in Sierra Leone stemmed from two genetically distinct EBOV Makona lineages, bboliaved to have been introduced from Guinea, One ofthese ln- ‘2ages (SL) was more closely related to the then-avatlable three Guinean genomes (two to five mutations) than the second line ‘age S12}, which was characterized by four additional mutations. ‘This finding suggested that SL2 had evolved from SL1 some ‘months before it was observed in Sierra Leone. A third lineage (GL3), derived from SL2, emerged in mid-June 2014. SL3 difers from SL2 by single mutation at postion 10.218, fst found asan intrahost variant (polymorphism within one individual) at a low frequency. SL3 became the most prevalent lineage in Sierra Leone during the fist 3 weeks of the outbreak there, with SLI disappearing soon after the appearance of SL3. The SL3- 15% frequency in eight diferent samples (Figure S20), a ‘much higher frequency than expected the change represented ‘de novo mutation in each sample, In summary, we conclude that a combination of human-to- human transmission and recurrent mutationsis likely responsible for the ISNV pattem observed in Figure 2A. This hypothesis is supported by the ISNV at position 18,911: samples containing this variant often cluster on the phylogenetic tre (Figure 28), although more isolated samples may represent separate muta- tion events. More general, pairs of samples that share an 'SNV are typically located near one another phylogenetically: these pairs are separated by an average of 0.16 years of (Col 161, 1518-1526, June 18, 2015 ©2015 The Authors. 1519Cell > patients with shared intrahost variants Jun ul Aug Synonymous | Nonsynorymous May Jun July Aug ‘evolution, whereas random pairs are separated by an average of 0130 years (p < 10°, randomization tes), These results suggest transmission of iSNVs in at least some cases and therefore ‘suggest thatthe transmission bottleneck is wide enough to faci tate the transmission of low- or intermadiate-frequency variants between hosts. Viral Evolution during a Prolonged EVD Epidemic We previously reported that new mutations accumulated more rapidly in the viral population early in the outbreak than over the long-term inthe reservoir (Gre et al, 20'4), We hypothesized then that the higher rate early in the outbreak resulted trom incomplete purifying selection—that is, we were detecting tran- sient nonsynonymous variants that would later be removed by Purifying selection (Pybus et al., 2007; Bedford et al., 2017). ‘The observed evolutionary rates thus not an estimate of the un- 1820 Coll 161, 1516-1526, June 18, 2015 ©2015 The Authors Wi tencoding 0 Figure 2. Evidence for Host-to-Host Trans mmission of Multiple Ebola Virus Makona 7 Genomes 1) Cart intahost variants (SHV) appear in samples throughout he 2019-201 EVD epidemic Siggesing tat SM can be ranemite betweon patents Varants share between two of more amples are shown as rows of camected pots: ‘each row is 2 genome postion (oered by poston ang the genome, top to bottom), ane ‘ech pont inaeats he presence oftheiSV ina (© Pryogenctc placement of deed toes at ‘geneme postion 18.917 implies both repeated ‘of recurent mutation, Colored tps ae sized ac- {cording to tequency of SMW at poston 18911 Tips with smal black pos ae those wh SV cals at any positon: other tps reteset samples wih ne SNV calls. Tis figure shows ont the orton ofthe tee rlvant fo tis anaes; ge branches wthno SNPs ors a positon 18971 re ot sou, Soe aio Fire 0. derlying mutation rate since some delete ‘ious mutations are purged by selection before they can be detected. But neither isitan estimate ofthe long-term substitu tion rate since other delaterious mutations have not been eliminated by selection at the time of analysis. We hypothesized that the EBOV Makona evolutionary rate ‘would decline folowing the addition of genomes covering a longer evolutionary timescale, Such a decline is well charac- terized in members of other species (Duchene et al., 2014; Ho et al, 2005). With the present data set, we were able to examine the evolution of the virus ‘over a longer time period. We found that the mast probable estimated evolutionary rate of EBOV Makona is indeed markedly lower (mean posterior rate = 1.25 x 10 * substitutions per site Per year) and is closer to the long-term rate than tothe rate est- mated early inthe outbreak (Figures 9A and S:). How purifying selection acts at cifferent timescales can also be saan in the distribution of mutations in the EBV Makona ‘genealogy. Deloterious mutations are more likely to result in ‘ransmission-impaired viruses and dead-end infections and may therefore oniy be present in individual patients. Mutations Unique to individual patients are those that occur on the external branches ofthe phylogenetic tree, whereas intemal branch mu- tations are those present in multiple samples in our data set ‘Thus, in the model of incomplete puritying selection, we expect ‘external branches to be characterized by ahigher rate of nonsy- honymous substitution than internal branches: in the latter, ‘selection has had more opportunity to fiter out deleterious mu tants, Internal branches, by definition, have produced muitiple re> o posta danty err deny " 401 18. descendent ineages and are thus less likely to include mutations: with fitness costs. To test this hypothesis, we estimated the rhumbers of nonsynonymous and synonymous changes on the vius genealogy and recovered their accumulation rates (Fia- lure 8B). Nonsynonymous mutations indeed occurred at lower frequency on intemal than on external branches, suggesting ‘that most are removed by puntying selection because of their ‘fitness costs and hence represent evolutionary dead ends. Syn fonymous mutations, which likely have less impact on fitness, occurred at more comparable frequencies on intemal and extemal branches, “The relationship between the effectiveness of purifying selec ‘on and its dutation is also apparent in the overall pattern of ‘nonsynonymous mutations in our data set. Selection fits the. accumulation of coding variants in the EBOV genome (Figures 8G and 4A), Nonsynonymous mutations, which are more tkely ‘to be deleterious, make up a decreasing fraction of coding mu tations as we analyze longer timescales: intrahost variants > in- dividual patients (external branches) > multiple patients (internal branches) > between outbreaks. The fraction seen between out- breaks represents the effect of long periods of evolution in the: Unknown EBOV reservoir. AS selection acts to remove delete- rious alleles over time, fewer nonsynonymous mutations can be detected. This pattern holds true across the EBOV Makona ‘genome (Figure “A Possible Host Effects on the Viral Genome ‘Although we observe less constraint on nonsynonymous changes during the 2013-2018 epidemic than between out- breaks, one anomaly is the genomic sequence encoding the Imucin-ike domain of the EBOV glycoprotein (GP), for which Wwe observe more nonsynonymous substitutions than expected Under neutrality, both within and between EVD outbreaks. Selec- Figure 8. Ebola Virus Evolution during a Prolonged EVD Epidemic 1 Estimats of EBOV evolutionary ats at tree ‘imescalee: decades (ylow, al krown EVD ca brake), monthe ue, Base © Gir» Pat ad ook ot: Baza + Gi). 1B) Purtying selacton. We estimated non 'symorymous ved and smorymous (us subst {ton rales on extemal (nique to an iol, potental dead en) and ins shared by mat le oles, evcerce of hunan-o-human tans Imsen) branches. Nonsynonjmous mutations fccumulte faster on extemal ranches than on Internal ances For synonymous mutations. the ference betwoen extemal and intemal ranches Isles pronounces, (@) Emtcrmort for nonsynonymeus mutatons shorter timescales. Invahost (al variants that appear within a ergo host at less than 70DK ‘tequenc uriaueirterost SNPs fren erat ne inh shared interact (SNP food ‘0 oF more individ shared botwoon EVD cutee (nema branch SNP on 3 Eatwoan ‘xtra | Soo aso Fe SA, tive pressure acting on a region can be estimated with the standard statistic dy/ds, which has an expected value of 1.0, for neutral evolution and less than 1 for purifying selection; in the mucin-ke domain, the mean posterior di/ds within this outbreak is 4.74, and between outbreaks is 1.44 (Figure 4A) GP is the only surface-exposed viral protein on EBOV virions, {and as such, itis the primary target of antibodies (Mun et al, 20). This finding therefore raises the possibilty that antibodies ‘might be driving diversifying selection and rapid evolution in this region. This observation is basad on a very small number of ub- slitutions (eight nonsynonymous and four synonymous within the outbreak), however, and isnot statistically significant (posterior probability that dy/ds is elevated within-outbreak = 92.996); the situation should be clarified as more sequencing becomes avall- able. If diversifying selection is occurring here, then the observed ‘changes are very unlikely to represent population-level selection for transmission among humans; this would only occur if previ ously infected individuals were frequently being exposed 10 new infections, Instead, we hypothesize that these changes. represent within-host selection for EBOV to escape a developing humoral immune response. ‘Totest the hypothesis that antibodies dive diversifying selec tion of GP, we looked for enrichment of mutations within B cell epitopes within that protein. Effective humoral immunity de- pends on antibody binding to specific B cel epitopes (Becquart tal, 2014; Murin eta, 2014). Using experimentally determined 8 cell epitopes obtained from the Virus Pathogen Database and ‘Analysis Resource (VIPR; Picket! ot a, 2012), we found that non- ‘synonymous mutations in GP do indeed acer mare frequently in ‘epitopes than expected by chanca (Figure 4B), This correlation ‘supports the hypothesis that humoral immunity exerts selective pressure onthe viru, driving immune evasion via accumulation ‘of nonsynonymous mutations within GP B cell epitopes. (Col 161, 1516-1526, June 18,2015 ©2015 The Authors. 1521ie) : Dd. e F | : Hit | il wl inl (Cand F Bvated T0-Crato pene wide but arelimtadto a subset of sequences. Accumulation of mutatenincreasestneary wit ie. However, Som Figure 4. Evidence for Host Effects on Ebola Virus Makona Evolution (9 Nonsyrenymous varants ar enviched in he rcrlke domain of GP. Estimates of oot) (aka loge) por coding sequence win the ister Aican EVD oxbreak ef) and betweon EVD outbreaks (ight) demonstate gene-specte pater of natural selector (®) Nonsynonymous varants are erviched In 8 call eptopes of GP. We calculted the rac. tins of nonsyeonymous (NS) and synonymous (9) consensus SNPs and nanost variants (SNV5) within experinertaty determined 8 ee eptopes (data tom MPR: Prk tal, 2012, Dotted tne ropresents the tacton of GP amine acts in VPA epitopes. Nonsynonymeus ‘SNPs (p = 0.004) and ISNVe p = 0.037) n GP cea more frequent in eptopes than expocted by chance foiled exact binoral tet [Numbers inate fraction ofeach variant type within GP eptope regions. Err bars represent ioral sampling intoval (©) Local enrichment of T40-C mutations win (GP Bcol epitopes. Wo observed five sequences with short -srochos (<200 nucleotides) of Concentrated T-1o-C mettions. OF Da to 9 ‘quences, Io (ser oro, samples 20141582 land G5119-1) contain sttehar of TC SNPS (ol points wtin GP epitopes (itt Bue bars Aaciionaly, wo obsone a T--C mutation at amin ack postion 485 ue dann) noe Samples (one oun here, GA955.1), whieh there compet conserved amen mame fa atau species (il ta, 2012) (0) Goneme-wice ncrease in T-6-C mutations. We oteerve more T-o-C tanatione within the 2013-2018 outbreak than ary ether wanton, ater coring fr nucleate conta Eso bars represent nomial samping intra Indveval samples stow mere genetic dance than expected based on sample dete. Samples with sor stsches of T-o-C mutations (orange) show & ‘gna enichmentofTo-C mutations, a expected. Excdng these samples, the top 5% of samples genetic distance (yellow lack caized stretches ‘tail show moderate enrichment of-o-C mutations genome wide, The bttom 95% of samples sige) sow no enickment of -1o-C mutton Ero are represan bisomalsanging itera Visual inspection identitied a subset of sequences that are ‘more likely to contain B cell escape variants (Figure 4C). In particular, three sequences (e.g., G4955.1) had a threonine-to- alanine mutation at GP amino acid position 485, a conserved threonine that is required for in vivo protection by the 14G7 ant body (Ola etal, 2012). Additionally, two sequences had short stretches of T-to-C mutations in GP (four or more T-to-C muta- tions within a 200 nucleotide region; Figure 4C), both of which ‘occur within 8 call epitopes. ‘Similar patterns of excess T-to-C mutations within short re- ions were also observed by Tong etal (2075). In our data set (0f318 genomes; five possessed obvious stretches ofT-t0-C mu tations within short regions. We also tested more broadly \whather excessive T-to-C mutations occurred in all sequences and found a significant enrichment of T-to-C transitions relative toallother types of transitions (Figure 4D). To determine whether viral sequence divergence is related to T-to-C transition enrich- ‘ment, we compared relative T-to-C transition rates in sequences 1822 Coll 161, 1516-1526, June 18, 2015 ©2015 The Authors with stretches of T-0-C mutations (n = 5) to the top 5% of remaining sequences by sequence divergence (n the bottom 95% of sequences (n = 298) (Figure AE}. While the ‘sequences with T-1o-C stretches showed the strongest T-to-C ‘enrichment, we found moderate enrichment of T-1o-C transitions in the 59% most divergent sequences, DISCUSSION ‘Our findings from 282 EBOV Makona genomes sampled in Sierra Leone over 7 months during the 2013-2015 EVD outbreak in Western Africa demonstrate the value of continued sequencing throughout an epidemic. We tracked the movement of EBOV ‘throughout Sierra Leone and determined the frequency of EBOV movement into and out of that country. Although its not Unlikely that the virus continued to cross the national borders ‘of Sierra Leone throughout the epidemic, these observations ‘suggest that, atleast in late 2014, cross-border introductions‘wore not an important factorin the development ofthe epidemic. \We were unable, however, to draw any conclusions about export to Guinea since few EBOV sequences from there are currently avaliable. ‘The sequence data display EBOV Makona evolution in the context of prolonged human-to-human transmission and pro- Vide an updated view of genomic diversity. Based on the rates: ‘of nonsynonymous and synonymous changes that are shared fF are unique to an individual host, we concluded that purliying selection becomes increasingly effective over time, as it has ‘more opportunity to remove deleterious mutants. ‘While the effects of purifying selection inthis extended EVD outbreak are clear, these evolutionary changes do not imply that positive selection or adaptation to humans are occurring Rather, the data suggest that evolutionary changes over tine through natural selection are sufficient to remove newly arisen alleles that are less fit in the human environment. To date, no Published study has found experimental evidence of selection {oraleles beneficial tothe virus within the current outbreak. It is important to recognize, however, thatthe long-term hu- ‘man-to-human transmission observed during the 2013-2015 EVD outbreak is historically unique for EBOV. At the beginning of each EVD outbreak, EBOV enters the human population with litle or ne genetic diversity In the case of the current EVD outbreak, EBOV has now maintained fitness while expanding ‘cross a much larger space of genetic diversity than in previous. EVD outbreaks, the largest of which comprised only 318 human infections. This degree of diversity wil undoubtedly affect re- searchers’ ongoing efforts to develop or improve candidate di- agnostics, vaccines, and therapeutics for EVD, many of which are targeting EBOV sequences directly (PCR, nucieic-acid based therapeutics) or indirectly (antibody cocktails). “The mucin-Ike domain of the EBOV glycoprotein, in contrast to the rest of the EBOV genome, appeared to be under dversi- {ying selection based on a high ratio of nonsynonymous-to-syn- ‘onymous mutations. While not statistically significant because of the small number of SNPS in the region, our observation is in agreement with many previous studies (Sanchez et al, 1998; Wertheim and Worobey, 2009). As the EBOV GP, especially the mucin-ike domain, is the target of many antibodies, a plau- sible hypothesis is that the humoral immune response exerts selective pressure on GP, resulting in an accumulation of nonsy- Ronymous mutations. In support of this hypothesis, regions of GP corresponding o experimentally determined B call epitopes. are significantly enriched in nonsynonymous, but notin synony- ‘mous, variants. There are two important caveats to this analysis: (1) these epitopes are determined in vito and therefore may not be epitopesin vivo ifthey are not immunodominant, and (2) there. s no experimental evidence to suggest that the majority of observed variants disrupt antibody binging to these epitopes. ‘While further experimental testing is required to validate an immune evasion hypothesis, we have highlighted a few prime candidates to consider. Genomes from three samples share a threenine-to-alanine mutation at GP amino acid position 485, a position that is conserved among all members of the Ebolavius ‘genus. This positions indispensable for binding ofthe protective antibody 1467 (Ola eta, 2012); the observed variant at tis site may therefore be the result of escape from antibody-mediated selection. Additionally, two samples each possess. multiple ‘mutations within a single experimental 8 cell epitope in GP, which are likely to evade antibody recognition if those regions are relevant epitopes in vivo. Intriguingly, the two samples with multiple mutations within a single B cel epitope each possess distinct short strate ittered with T-1o-C transitions, a phenomenon also observed in Tong tal. 2075). Excessive T-0-C and A-to-G mutation of virus ge- homes has been observed previously as a result of adenosine deaminases acting on RNA (ADARSs; Gélinas et al, 2011; Zahn {etal 2007; Carpenter tal, 2008), When acting on viral genomic INA, ADARs cause a pattem of excess A-to-G transitions that are represented by T-1o-C transitions in our data set. These tran- sitions are known to occur either promiscuously within 200, ‘hucleotide stretches or in a sequence-specific manner; there- fore, we investigated both possibiitias. While only five of the 818 sequences in our data set contained obvious T-1o-C stretches, we showed that the top 5% of sequences by sequence divergence, excluding the five sequences with T-to- C stretches, wore also moderately enriched for T-o-C transi- tions across the genome. The remaining 95% of sequences appeared to show no enrichment. We do not know whether this phenomenon is caused by ADAR acting upon genomic INA, as we cannot exclude the possibity of bias by the EBOV FINA polymerase or other effects, Additonaly tis yet unclear whether these T-to-C mutations have an anti-viral or other effect (on viral fitness. These questions open avenues of research into ‘molecular mechanisms shaping EBOV evolution. ‘The resuits of some ofthe specific genome analysis methods that we introduced here, while promising, wil require denser EBOV genome sampling to yield sufficient information to influ- fence the EVD outbreak response, Among these methods is, transmission analysis, which could prove valuable for improved Understanding of hospital-based transmissions and therefore for Improved infection control. Inference of the ancestral genetic slate is often straightforward, with clear pattems of new varia- tions layering on previously existing variations; viruses that appear to be descended from others in the same data set are ‘separated only by new mutations that are seen nowhere else in the data set. Ths kind of genetic relationship does not guarantee a transmission relationship between two pationts since many vi- ruses can share identical genomes. However, since viruses with Identical genomes are often epidemiclogically related (Gire et al. 2014), we can infer that viruses that appear to descend from other viruses in our data set are either in or epidemiologically Close to the same transmission chain. Unfortunately, long delays of shipping samples from the field and required changes to the EBOV inactivation protocol caused severe degradation of many samples, which pre- vented identification of variants and transmission analysis This loss should serve as a reminder that standardized and ‘optimized protocols for sample collection, virus deactivation, and shipment are crucial for a rapid worldwide response 10 ‘any new infectious disease outbreak. An important future research effort wil be aimed at understanding which certified EVD sample deactivation protocols are best suited for high- {quality genomic sequencing. Complications with sample ship- ment also emphasize the need for establishing in-country (Col 161, 1518-1526, June 18, 2015 ©2015 The Authors. 1523 Cellie) ‘sequencing capabilities either before or at the onset of future EVD outbreaks (Folarin et al, 2014), Beyond coordinated field and experimental responses, a cul- ture of rapid data sharing is critical for teams around the world 10 have the best current information about a circulating vieus oF ‘ongoing disease (Yozwiak etal, 2019) In ight ofthis need, we released all data discussed in this paper publicly as they were generated, beginning in December 2014, well in advance of our ‘own analysis. We have previously deseribed our high-depth Sequencing protocols (vialranga et al, 2014), and we are also ‘now making available our computational analysis pipeline, in the hope that they will assist the many laboratories engaged in viral, ‘genomic research, As more EBOV genomic data become avali- able, in particular for poorly covered Liberia and Guinea, the scien- tiie Community can togather tain a broader picture of transmis- sion and evolution of EBOV Makona during the EVD epidemic. ‘Sample Preparation from Kenema Government Hospital ‘ns sty included 575 blood sare om 8 pater with conemed EVD from sue 16 trough Septmoe 28, 2014 by KGH lboratry stat. cies samples were nacveted using QIAGEN AVL and ethanol the KGH aber tory pero sipping out of te county ‘Sample Proparation from CDC Bo Laboratory Ths study ncuod 98 bio samples tom 08 patnts with cnfmed EVD from August 20,2014 tough Januay 10,2018 by CDC laboratory staf st tioned in Bo, Sia Leone, Clea species fom the CDC Bo laberatony In Serra Leone were shipped to and stored at tha Vial Special Pathogens Branch BSL laboratory atthe COC in Atanta, GA. Samples wore nactveted land RNA was exacted using the MagMIAX Pathogen RNADNA isolation Kt (iverogen and BesdRetever(nregen. Nn-inectious ANA was tested with DNase | Riase-ee (Roche) prior to shipment tothe Broad nse High-Throughput Sequencing Hos ribosomal and camer BOMPA] RNA depletion, randomly primed cONA sinthests, Nextra XT lary constuction, and 101-bp parad-end Raina Sequencing were peiomed as cescrbed previously (Soot a, 2014 Ma> tranon et a 2019 [Ebola Virus Makona Genome Assembly and Analysis EBOV Makona genomes wor assomd tom high troughput saqueneing ata using an Updated Dofomaiee pipeino based on cur preva, ‘escrbox mathods (Sree 2014 Matranga 201) Othe ctected ‘samples, 150 KGH and 82 COG samples had sulcnt EBOY genome Siaqencing coverage fer high-quality do rovo gnome assembly. Further esorpton ofthe pele ean be found Inthe Sunplemen!Experinonts Process, (Our Liu-based software ppdine Is publ avalible at his/ishub. combosaiatuta/val-nas (Park ta, 2019. Tis pane nts com- ‘mard-inotaos foreach ofthe above stops ardoptional Saakemakeworkows (Késterand Ratmare 2012}toautomatethom other sequently orn para “he assembly Paine i aso aval via the DNAnews cloud platform. ANA patreciend reads from ether HSeq or MiSeq insruments flumea) can bo securely uploaded fn FASTO or BAM format and processod tough the pipalne wang graphical and commands itraces. Instructions for the cloud anayse pipeline are avalable at htps//otnub.convthanows! vrata ‘Genomic Epidemiology of Ebola Virus Makona, “Tho flowing publ avalable EBOV Maker genomes tom cutsde ot Sia sone donot cary the SL3-derued allele at poston 10.278 26 ainda = romes fom Libera @5 fom Kuneiman sl. 2015, one fom GerBar 1824. Call 161, 1516-1526, June 18, 2015 ©2015 The Authors 178598.) anda four avaliable genomes om Mal Hoenn eta, 2015) ‘A medaning haplotype network was constucted ln PopART version 1L72 ta/popart ota so), Due tothe presence of missing data, 1462 ses (7.936 of total genom) were excluded trom the anal: these ates included 6 sts wih vaialty among lates (10.0% of al variable ates) “To reconstruct tho EBOV Makona tansmson story within Sa Looe, ‘we grouped sampies into sets of one or moe gens inten runes based on ther consensus sequences. Wo then Mend reitonshps between these groups, praressing fem the Guinean reerence genome (4600546 2) and ecina wih rine veuses sampled in Freetonn (ight rom ‘ur KGH and COC cohorts an one sequenced ay, Intrahost Variant Analysis Fu tis of he ientifeaton and caing of intabost arate (SAWS) are vation the Supiomrtal Exparimera!Procosues; SAV els and ana yes arava in Oss. Evouonary stances between pas of phyog- 2y tos were computed from the postr saree of wees produced by Bayesian evulonayaralytsby samping wees BEAST) [xurnong et al. 2012) analys. Ts calculation iterates acoss plogenstc unceanty ‘nd produces temporal eyoldionsy dstance between pyogeny tips. We ‘sed this distance matns 1 caeulate the average distance between pars of Pvlogory tps at share an SAV and compared the resul tothe average ds- tance between random pars ofp. We calcuatd ap valu for tho booed ‘vege distance by conducing a randemzation et In each random rep ‘ate, we sampled the cam dstrbuton of SNV possessing ps as booed Inthe empiical data and calculate no average distance between these pas cp. We cause ap value by comparing the enpcal mean stance to the mean distances observed ovr 10.00 random epicates {GP B Coll Epitope Analysis Data were obtained om the NIAID Vue Pathogen Database and Anais Resource PR) enn tough he web ae a p/w vob cro eke ta, 2012). Asmostof he epepesin the database are based the Mayings ‘eference sri, we mappedalB celleptopes aginst the Guinean reterence ‘an (GenBank KJ6609462) nel removed al eptopes that no longer matched perfec, leaving 40 B call epitopes Overapoing eptopes were -merged and nonsynonymous snd synonymous SNPs and SAWS were scored ‘win or cutee of epitope egons Sigiicance was deere by two {ted ino tea ith = 0.05, wh the nl Iypoesa thet variants woul ‘occur neptoperegens fGP by chance uth peda 172/875, wchsthe traction of GP esidues GP whtin cal eptpes, Molecular Evolution “Three datasets ware constructedto represent hrestinescalsco genetic sur velance ct EBOV Makona For suvetiance betveen EVD euros, 62 pu ey avatable sequences rpresert th every of EBOV saad over ong periods oftime:thesa sequences che the ist recorded 7976 EVD oor ‘ns oer EVD outbreak an excl one outoreak occuring inthe Damo: ‘atc Repubictthe Congo n2014, WealeoincucedEBOV ganar rgrent “sequences rom possibly nectes great ape carcasses and runvorus ats Fourteen sequences rom Westem Area were chosento represent tna corert 2010-2015 EVD abrenk Fer survllance ofthe ary oueeak, Bt saquences (Gaze tal, 2014; Greta, 2013} were reanzed epresenting the caries ‘pcemilogesly elevant and pubey atatable sequences. For suveance ‘line prolonged ape, 232 EBOV genomes reported nee werecompined ‘wah tie sequences wom repatated heahcare workers (UKT, UR2, UG, Int, GEN and te 81 Sequences tom he ay outbreak dela Set ‘Aaciyes of rates, pyogenes, and evaluten were perorred en al vee ta sets nBEAST (numvnond.! a 2012) Fuldetasenthe model ds ‘ameter are avaiable the Supplemtal Exeermeri Procedures. Al BEAST inputs, ctpus, and anayss srpts ae avalabe in ats 52 ACCESSION NUMBERS. Genome assemblies, annotations, an raw ras are svasle at NCBI on “Gengank and SRA using he folowing oProoctIDs: PRINAZS? 97 (sarpestrom Kenama Government Hospt) and PRUNA2ES985 (eumples fiom CDC Bo Lab. Note hat PRUNAZS?197 aloo inches a previously publaned dala from Gee a 2019. Suplementlnlrnstion meals Supplemental Experimental Procaces, tou tae, wo abies, and two data les an ean Be foun wth the ae nine a ip do o/10.106/ 088.2018 05 007 AUTHOR CONTRIBUTIONS. ‘The conibatons af each authr ate an extensive to itn eta. Bu song eta ee andl our suhors, AG. ar FG. coletedsampis. AG and SLM.W. processesampies for sequencing, OP, GD, SV, SLM.W.and [AR snaijeed sequence dts, DP, GD, SW, SLMM., US. AR, and PCS. woe te paper US. AR, RF. and PS. jorysuncised he work ACKNOWLEDGMENTS We thank KG stat who dec of VD fnehicng Mt. Fons, A. Moigb A. Ko- ‘yma, M. Fulah, and SH, Khan), the Ofice ofthe Pesisant of Sera Leene [PraidentEKoroma, M. Jones. the Sera Leon Minty f Heath a San tation, the Kanna District Heath Management eam an the Kenora Lassa fever program for thir mental inthe EVD outbreak responce. We ‘ark Puble Heath Engh (UKE, UK2, UKS) IRCCS Lazzaro Spalinzan A), and tho Unsersy of Genova (SE!) for providing EBOV genome s0- ‘quences tom samples of EVD pats expat rm Sioa Lone. We thank the crvers, pts, phieotemiss,ron-governmestal omanzations, dsirict redial ofcers, and dstict savelance offers forthe hp wh sample ‘alban ardlogses in Sera sve. We want a espe thnk the Me ‘ns Sans Fronres MSF operation centers the continuing support ofthe US Cones for Disease Corto! and Prevention (COO) aboratory in Bo, Sera Lone andthe Word Heath Organization WHO) fr ter supper ofthe re ‘zing COC brary operation n Kenems, Sins Leone. “Tis work was supported by European Un rant FP7/2007-2018278433- PREDEMICS and Ewepean Reseach Counc grat 260864 (AR); Naural EnyonmontResoareh Councl grant DYS730X (8.0) US# GM 1274 (TB NM rant GM080177 (WN grant 1UO1HGOO7480.01 (CH No ‘tonal Seance Foundation Grecusta Resaorch Fallwehip Grant No. OGE 1144182 (AE): the National Heath anc Medical Research Counc, Austral CH the Defence Treat Reducten Agency (USAMRID, NINN Uiaai11081@ (Broad ist), tho Bland Neinda Getes Foundation (0PP' 123407 readin) and MIND HHSN272200000040C Hare lone. Ths work was funded, in part, through tte Memorial nett ris contracture US National nse of Alergy andinfactious Dasarss INIA) uncer contact numer HHSNZ722007000181, Subcontractors to Bat {lo Memoria insite who perered this work are J HK, an employee of “Tunnel Goverment Senvces he. RIF. cofounder Zigen Loe “The Vi Pltegen Database and Analysis Resource (IPRs been wholy tuned with federal us rom the ational atte of Alay an ietous Diseases, Nations sites of Heath, Department of Health ana Hun Ser ‘ce, under contact sumer HHSN27220 14000280, “Ti paper was shored a Authocea, nd et hor is eal here: pater shore conser 0734/1957 “The content otha publication doesnot necessary reflect he Views or pal- ‘eins line US Departmen of Health ad ran Servos (Centers or see Control and Prevention, Hatona nates of Heath) or tbe US Ay Receive: May 18,2015, Revised: May 25,2015, Accepted: Jone 1 2015 Pbk: June 18,2015 REFERENCES ‘Aaon, 8. 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