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Published in final edited form as: Virology. 2016 Jun 29;496:237–243. doi: 10.1016/j.virol.2016.06.011

Virus fitness differences observed between two naturally occurring isolates of Ebola virus Makona variant using a reverse genetics approach

César G Albariño 1,*, Lisa Wiggleton Guerrero 1, Ayan K Chakrabarti 1, Markus H Kainulainen 1, Shannon LM Whitmer 1, Stephen R Welch 1, Stuart T Nichol 1
PMCID: PMC6745713  NIHMSID: NIHMS917928  PMID: 27366976

Abstract

During the large outbreak of Ebola virus disease that occurred in Western Africa from late 2013 to early 2016, several hundred Ebola virus (EBOV) genomes have been sequenced and the virus genetic drift analyzed. In a previous report, we described an efficient reverse genetics system designed to generate recombinant EBOV based on a Makona variant isolate obtained in 2014. Using this system, we characterized the replication and fitness of 2 isolates of the Makona variant. These virus isolates are nearly identical at the genetic level, but have single amino acid differences in the VP30 and L proteins. The potential effects of these differences were tested using minigenomes and recombinant viruses. The results obtained with this approach are consistent with the role of VP30 and L as components of the EBOV RNA replication machinery. Moreover, the 2 isolates exhibited clear fitness differences in competitive growth assays.

Keywords: Ebola virus, West Africa outbreak, Makona variant, Recombinant virus, Reverse genetics

1. Introduction

Several members of the viral family Filoviridae, including Ebola (EBOV), Sudan, Bundibugyo, Marburg, and Ravn viruses, have caused sporadic outbreaks of hemorrhagic fevers in central African countries. Another filovirus, Tai Forest virus (TAFV), was responsible for only one case of human disease, while Reston virus (RESTV) and Lloviu virus have not been associated with human disease to date (Feldmann et al., 2013; Leroy et al., 2011; Rougeron et al., 2015).

Filovirus particles are enveloped and carry a single-stranded, negative-sense RNA genome of approximately 19 kb (Feldmann et al., 2013). The filoviral genome encodes 7 genes: nucleoprotein (NP), VP35, VP40, GP, VP30, VP24, and the RNA polymerase L; these genes are transcribed sequentially from the 3’ end of the viral genome (Mühlberger, 2007) (Fig. 1(A)).

Fig. 1.

Fig. 1.

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(A) Schematic representation of the Ebola virus (EBOV) genome depicted in the viral complementary sense, with the open reading frames of the 7 genes depicted in the 5′ to 3′ orientation. (B) Growth kinetics of wild-type isolated viruses. Huh7 cells were infected at multiplicity of infection (MOI) of 0.1 with human clinical sample isolates of the Makona variant (Mak-1, GenBank accession no. KP178538; Mak-2, GenBank accession no. KP240932). Growth kinetics were assessed by determining viral titers in cell supernatants using a standard TCID50 assay. (C) Sequence comparisons of full-length genomes of Mak-1 and Mak-2 isolates. The positions of the nucleotide changes, gene locations, and amino acid changes are shown.

Caused by EBOV, an outbreak of Ebola virus disease (EVD) of unprecedented magnitude started in late 2013 in Guinea and quickly spread throughout the country and to the neighboring countries of Liberia and Sierra Leone during 2013–2016 (Baize et al., 2014; Gatherer, 2014). A few cases, mostly imported, were also detected in Nigeria, Senegal, and Mali, and eventually in Europe and the United States (Centers for Disease Control and Prevention, 2015; WHO, 2016). A total of 28,608 cases and 11,305 deaths, mainly from Western Africa, were recorded as of March 2016 (Centers for Disease Control and Prevention, 2016).

Because of the large extent of the 2013–2016 outbreak, several hundred EBOV isolate genomes were characterized (Carroll et al., 2015; Gire et al., 2014; Ladner et al., 2015; Tong et al., 2015) after the initial description of the new circulating Makona EBOV variant (Baize et al., 2014; Kuhn et al., 2014). Genetic analysis of the sequences obtained during the outbreak allowed inference of the evolutionary rate and genetic drift that appear to have occurred after the initial spill-over event from the yet-unknown natural reservoir into the human population. The results suggested that the genetic drift was probably due to multiple events of human-to-human transmission (Hoenen et al., 2015; Kugelman et al., 2015a, 2015b; Park et al., 2015).

Besides the potential role of genetic drift in the evolution of EBOV, the relevance of sequence variability to the viral phenotype clearly deserves further research. We previously compared the growth characteristics of 2 wild-type EBOV isolates (Albariño et al., 2015), one from the Yambuku variant (Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga) and one from the Makona variant (Ebola virus/H.sapiens-wt/LBR/2014/Makona-201403007); we found no significant growth differences when using Vero-E6 and Huh7 cells. Moreover, no significant differences in VP35 and VP24 activities were found between the Mayinga isolate and 3 Makona isolates when using a minigenome assay and an interferon inhibition assay (Dunham et al., 2015).

In this report, we analyzed the in vitro characteristics of the 2 Makona virus isolates, Mak-1 (Ebola virus/H.sapiens-wt/LBR/2014/Makona-201403007) and Mak-2 (Ebola virus/H.sapiens-wt/LBR/2014/Makona-201403261), that were described elsewhere (Whitmer et al., manuscript in preparation). Mak-1 was isolated from a clinical sample collected during the acute phase of EVD of a patient admitted in a hospital in the United States after a medical evacuation from Liberia in 2014 (Lyon et al., 2014). Similarly, Mak-2 was isolated during the acute phase of EVD of another patient that travelled from Liberia to the United States in 2014 (Chevalier et al., 2014; Liddell et al., 2015). This first patient eventually recovered from EVD, while the second succumbed to the disease and infected 2 health care workers, who later recovered. Due to the different clinical outcomes associated with these isolates, we sought to determine their basic phenotype when propagated in-vitro.

2. Results and discussion

Although Mak-1 and Mak-2 exhibited identical growth kinetics in Vero-E6 cells (Fig. 1(B)), they possessed interesting genetic differences from each other. As shown in Fig. 1(C), comparing the sequences of the viral genomes showed that the isolates differed from each other by only 9 nucleotides. Two nucleotide differences were located in the untranslated region of the NP and VP30 genes, and 5 represented silent changes, but 2 differences resulted in amino acid (aa) changes in VP30 and L proteins. Although alterations in the untranslated regions may affect the expression of adjacent genes in EBOV and Marburg virus genomes (Alonso and Patterson, 2013; Brauburger et al., 2015), we focused our analysis on the nonsynonymous mutations in the VP30 and L genes because of their potential implications for transcription and replication.

Both VP30 and L are essential components of the polymerase complex and make specific contributions during transcription and replication (Biedenkopf et al., 2013; Martinez et al., 2008, 2011; Mühlberger, 2007). The aa difference in VP30 of Mak-2 was particularly interesting because it generated a new stop codon (W282stop) truncating the last 6 aa, as we and others have previously described (Kugelman et al., 2015b; Ladner et al., 2015; Whitmer et al., manuscript in preparation). This change in the C-terminal domain of VP30 could affect transcription and the general fitness of the Mak-2 isolate (Hartlieb et al., 2007).

In contrast to EBOV, RESTV has not been associated with EVD in humans, but was found in non-human primates imported from the Philippines into laboratories in USA (1989 and 1996) and Italy (1992) (Jahrling et al., 1990; Miranda et al., 1999; Rollin et al., 1999). Later, in 2008 and 2009, RESTV was also found in domestic swine in large farming operations in the Philippines (Barrette et al., 2009). Interestingly, 2 genomic sequences of RESTV isolated from pigs, but not from non-human primates, carry a 6 aa truncation in VP30 similar to what we found in EBOV Mak-2 (Suppl. Fig. 1A). More studies are needed to determine any phenotypic effects of this truncation.

The aa difference in Mak-2 L (G882S) is located in a block of aa that is highly conserved among different filoviruses (Suppl. Fig. 1B). It is also in proximity to one of the predicted motifs (motif E, positions 798–809) and the predicted catalytic site of filovirus polymerases (GDN741) (Jacome et al., 2015; Volchkov et al., 1999).

In order to test any independent contributions of the aa differences to the replication phenotypes of the 2 isolates, we first created a new EBOV minigenome system that drives the expression of Gaussia luciferase (gLuc) during RNA replication and transcription (Fig. 2(A)). In contrast to the system described in our previous report (Uebelhoer et al., 2014), which was a T7-driven minigenome plasmid based on the Mayinga isolate, the Pol I-driven minigenome and protein expression plasmids (pC-NP, pC-L, pC-VP35, and pC-VP30) used in this current study corresponded to those of Mak-1 (GenBank accession no. KP178538); these plasmids were considered wild-type (WT). We then modified these protein expression plasmids (Fig. 2(B)) to express the truncated VP30 protein (W282stop) and the modified L protein (G882S) seen in Mak-2; we also used an inactive L (Linac; GDN741AAA) as a negative control. As shown in Fig. 2(C), co-transfection of Huh7 cells with the WT minigenome and protein expression plasmids (NP, VP35, VP30, and L) yielded a robust gLuc signal, while using the Linac control did not. Interestingly, using the Mak-2 VP30 expression plasmid in the minigenome assay increased the gLuc signal by 19%, and using the Mak-2 L expression plasmid increased gLuc signal by 56%; no synergistic effect was observed when Mak-2 VP30 and L were used together (Fig. 2(C)). The expression level of WT and mutant proteins showed no significant differences (Suppl. Fig. 2A). Moreover, similar results were also observed when we tested the Mak-2 VP30 and L plasmids using our previously described Mayinga-based minigenome system (data not shown).

Fig. 2.

Fig. 2.

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(A) Schematic representation of EBOV minigenome. The gLuc reporter gene was cloned in the viral sense between 5′ and 3′ untranslated regions (UTR) of EBOV Mak-1 isolate (GenBank accession no. KP178538). Luciferase activity results from transcription and replication of the gLuc minigenome in the presence of the viral proteins L, NP, VP35, and VP30. (B) Protein expression plasmids were based on the EBOV Mak-1 isolate (GenBank accession no. KP178538) and codon-optimized for human cells. Wild-type and mutated plasmids are shown. (C) Minigenome activity. Huh7 cells were transfected with EBOV gLuc minigenome and support plasmids to express NP, VP35, VP30, and L. WT VP30 and L were replaced with plasmids bearing Mak-2 sequences where indicated (VP30mut and Lmut). Data are representative of 4 independent experiments, with means and standard error (error bars) of luciferase expression from 3 wells displayed in relative light units (RLUs).

To analyze the effects of the VP30 and L differences in the context of an infective virus, we used our previously described reverse genetics system (Albariño et al., 2015) to generate a recombinant Mak-1 derived virus (WT), 2 single mutant viruses carrying the VP30 (VP30mut) or L (Lmut) changes corresponding to the Mak-2 isolate, and a double mutant virus (VP30/Lmut) carrying both VP30 and L changes (Fig. 3(A)). Using our standard protocols, WT, VP30mut, Lmut, and VP30/Lmut expressing a reporter gene (ZsGreen) were successfully rescued in Huh7 cells with similar efficiency (Fig. 3(B)). The expression levels of ZsGreen were comparable in all recombinant viruses.

Fig. 3.

Fig. 3.

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(A) Schematic representation of recombinant EBOV genome expressing ZsGreen (ZsG). Genome of rEBOV expressing ZsG fusion protein (Albariño et al., 2015) is shown in viral complementary sense. The plasmid transcribing the full-length genome of the Mak-1 isolate (WT) was further modified to generate single (VP30mut, Lmut) or double mutants (VP30/Lmut). (B) Rescue of recombinant EBOV. WT and mutated EBOV were rescued in Huh7 cells using the full-length clones and codon-optimized support plasmids that express NP, VP35, VP30, and L. Supernatants from transfected cells were passaged twice in fresh Huh7 cells, and average titers were assessed by standard TCID50 assay. Efficiency of viral rescue is shown as the number of positive wells (pos)/total wells. (C) Growth kinetics of recombinant viruses. Huh7 cells were infected with WT, single (VP30mut or Lmut), or double mutant (VP30/Lmut) viruses at moi=0.1. Growth kinetics were assessed by determining viral titers in cell supernatants using a standard TCID50 assay. (D) Competitive fitness assay. WT and double mutant recombinant viruses were mixed ~2:1 and used to infect Huh7 cells in triplicate. Cell supernatants were collected, diluted, and passaged onto fresh Huh7 cells 9 times. Total RNA was extracted using standard protocols and sequenced by next generation sequencing. The average percentages of WT and double mutant virus in the supernatants are shown for each passage.

In agreement with the results obtained using the Mak-1 and Mak-2 isolates (Fig. 1(B)), the growth kinetics of the recombinant viruses in Vero-E6 cells did not differ significantly (Fig. 3(C)). Similar results were also obtained using a set of recombinant viruses without reporter genes (data not shown). Overall, these results suggested that the sequence differences did not generate major phenotypic differences when viruses were individually propagated in cell culture.

Although the WT and mutant recombinant viruses exhibited similar growth patterns in our previous experiments, we hypothesized that a virus with slightly enhanced replication efficiency would have a modest fitness advantage, and would increase in relative abundance with each successive passage during a competitive fitness assay. To test this possibility, we designed a competition assay with the recombinant WT and the doublemutant (VP30/Lmut) viruses. Since the mutated VP30 and L proteins were hypothesized to provide a growth advantage, Huh7 cells were co-infected using a WT:VP30/Lmut ratio of 2:1. Three to four days later, cell supernatants were harvested, diluted, and passaged onto fresh cell monolayers. During 9 consecutive passages, samples were obtained for RNA extraction, and virus sequences were analyzed using next-generation sequencing protocols. In order to minimize the possibility of random events during the initial infection, 3 independent infections were performed and subjected to the same passaging protocol.

As shown in Fig. 3(D), VP30/Lmut represented 36% of the total virus in the original infection mix. By passage 3, however, 66% of the total virus in the cell supernatants was VP30/Lmut, and by passages 5, 7, and 9, VP30/Lmut constituted 85%, 93%, and 96% of the total virus, respectively. This competitive growth advantage of the VP30/Lmut virus over the WT virus correlated with the increases in minigenome activity seen in the presence of the modified VP30 and L proteins. The variant detection analysis, including the number of reads, the coverage, and the frequency of nucleotide changes of the fitness assay is shown in Supplementary Table 1.

Although the recombinant viruses WT and VP30/Lmut differ in only 2 aa changes, the original clinical sample isolates, Mak-1 and Mak-2, had 7 additional non-coding differences spread throughout the viral genome (Fig. 1(C)). To examine the role of the aa changes in the background of the non-coding changes, we tested the original isolates Mak-1 and Mak-2 using a competitive growth assay (Supp. Fig. 2B). The Mak-2 isolate represented 17% of the total virus in passage 1, and increased to 31% and 40% of the total virus by passages 2 and 3, respectively. Based on these results, the possibility of a rather minor compensatory effect of the non-coding differences over the 2 aa changes in VP30 and L could not be discarded.

3. Conclusions

In this report, we describe the use of a standard reverse genetics approach to characterize 2 isolates of EBOV Makona with single aa differences in the VP30 and L proteins. Our results indicate that the aa changes in VP30 and L proteins corresponding to the Mak-2 isolate sequence moderately increased minigenome activity, and the recombinant virus containing both changes exhibited a fitness advantage over the WT virus in a competitive infection experiment. Further epidemiological studies and experimentation in animal models will be needed to establish the potential significance of these minor sequence differences in the genomes of 2 EBOV isolates obtained during the 2013–2016 EVD outbreak.

The precise and systematic reverse genetics approach described here also provides a template for analyzing other EBOV genetic and protein alterations that occurred during the extensive human-to-human passaging of the virus during this enormous Western African EVD outbreak. Such studies will provide important insights into EBOV evolution and the potential for virus change that could alter the public health impact and epidemic threat posed by this severe pathogen.

4. Materials and methods

4.1. Cell culture and biosafety

All work with recombinant viruses was performed in a biosafety level 4 (BSL-4) facility. Huh7 (human liver cells) and Vero-E6 (African green monkey kidney cells) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum and penicillin-streptomycin.

4.2. Plasmid construction

Support plasmids and gLuc minigenomes were modified from those described before (Uebelhoer et al., 2014). First, support plasmids pC-L, pC-NP, pC-VP35, and pC-VP30 were designed to express human codon-optimized synthetic genes corresponding to those of EBOV Makona isolate Mak-1 (Ebola virus/H.sapiens-wt/LBR/2014/Makona-201403007; GenBank acc. KP178538). The original pC-VP30 plasmid was also modified to express the truncated VP30 protein (W282stop) as in Mak-2, and the pC-L plasmid was modified to express a mutated active L (G882S; also as in Mak-2) or Linac (GDN741AAA). The minigenome was also based on Mak-1 isolate, and was cloned in genomic orientation and flanked by the Pol I promoter (5′ terminus) and Pol I terminator (3′ terminus) (Hoffmann et al., 2000). The expression of gLuc was driven by minigenome transcription and replication.

In addition, pC-L and pC-VP30 plasmids were further modified with an N-terminal V5 tag. The expression levels of WT and mutant proteins were examined by western blotting as described (Albariño et al., 2015).

WT and ZsGreen-containing full-length clones were derived from the Mak-1 isolate, and were described in our previous report (Albariño et al., 2015). The full-length clones were later modified by changing the codons at position 9354 and 14,224 to introduce the W282stop and the G882S aa changes (as seen in Mak-2) in the VP30 and L proteins, respectively. All full-length clones were sequenced to completion. Details regarding construction strategies of support expression plasmids and plasmids encoding the full-length clones are available upon request.

4.3. Minigenome experiments

Luciferase minigenome experiments were carried out using similar conditions as previously reported (Uebelhoer et al., 2014). Monolayers of Huh7 cells growing in 24-well plates were transfected with 0.75 μg gLuc minigenome, 0.5 μg pC-L or pC-Linac (negative control), 0.25 μg pC-NP, 0.025 μg pC-VP35, and 0.025 μg pC-VP30. To test the effects of the Mak-2 aa sequences, pC-VP30mut and pC-Lmut were used instead of the corresponding WT plasmids. All transfections were done in triplicate, and the complete experiment was repeated 4 independent times. Three days post transfection, 5 μL of supernatant were harvested, and luciferase activity was measured using manufacturer’s instructions (Renilla Luciferase Assay; Promega) in a multi-mode microplate reader (BioTek Synergy).

4.4. Rescue of infectious viruses

Rescue of recombinant viruses was performed in Huh7 cells as described previously (Albariño et al., 2015). Briefly, Huh7 cells grown in 12-well plates were transfected with 1 μg pEBOV, 0.5 μg pC-L, 0.5 μg pC-NP, 0.05 μg pC-VP35, 0.05 μg pC-VP30, and 1 μg of codon-optimized pC-T7. In order to rescue single or double mutant viruses (VP30mut, Lmut, or VP30/Lmut), WT pEBOV full-length clone was replaced by plasmids carrying the aa changes found in Mak-2 VP30 or Mak-2 L, or both. Supernatants from transfected cells were passaged twice in fresh monolayers of Huh7 cells, and the rescue events were confirmed by immunostaining. A 100% rescue efficiency (positive detection in 12 of 12 replica wells) was seen in all rescue experiments. All recombinant viruses were sequenced to completion, and the viral genomic sequences were identical to those of the full-length plasmids. Virus titers were determined by tissue culture infective dose 50 (TCID50) assay, and growth curves were performed in Huh7 cells as described previously (Albariño et al., 2015).

4.5. Competitive fitness assay

To assess the relative viral fitness of recombinant WT and double mutant recombinant EBOV, the 2 viruses were mixed in an approximate 2:1 ratio, based on respective titers. The mix was diluted in order to infect Huh7 cells in triplicate using a moi of 0.1. After 1 h of adsorption, the virus was removed, the cell monolayers were washed once with PBS, and complete media was added. Three to four days post infection, cell supernatants were collected, clarified by low-speed centrifugation, diluted 1:10, and used to infect fresh Huh7 cells. A total of 9 passages was performed over 32 days, while aliquots of undiluted cell supernatants were also collected for RNA extraction using standard protocols. Total RNA from cell supernatants was used for next-generation sequencing by a commercial service provider (Omega BioServices). Basically, the cDNA libraries were constructed using TruSeq Stranded mRNA kit (Illumina), following a modified version of the manufacturer’s protocol that skipped steps necessary for mRNA purification and for rRNA depletion. Sequencing was performed using the paired-end 2 × 100 chemistry on an Illumina HiSeq 2500 instrument.

4.6. Sequence analysis

Nucleotide and aa sequence alignments were generated using CLC Genomics Workbench. The same software package was used to perform the analysis of next-generation sequencing data, including read mapping, contig assembly, and variant detection.

Supplementary Material

Suppl.Fig.1
Suppl.Fig.2
Suppl.Fig.3

Acknowledgments

We thank Tatyana Klimova for editing this manuscript. This study was reviewed and approved by CDC’s Institutional Biosafety Committee.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Footnotes

Appendix A. Supplementary material

Supplementary data associated with this article can be found in the online version at doi:http://dx.doi.org/10.1016/j.virol.2016.06.011.

References

  1. Albariño CG, Wiggleton Guerrero L, Lo MK, Nichol ST, Towner JS, 2015. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein. Virology 484, 259–264. [DOI] [PubMed] [Google Scholar]
  2. Alonso JA, Patterson JL, 2013. Sequence variability in viral genome non-coding regions likely contribute to observed differences in viral replication amongst MARV strains. Virology 440, 51–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, Soropogui B, Sow MS, Keita S, De Clerck H, Tiffany A, Dominguez G, Loua M, Traore A, Kolie M, Malano ER, Heleze E, Bocquin A, Mely S, Raoul H, Caro V, Cadar D, Gabriel M, Pahlmann M, Tappe D, Schmidt-Chanasit J, Impouma B, Diallo AK, Formenty P, Van Herp M, Günther S, 2014. Emergence of Zaire Ebola virus disease in Guinea. N Engl. J. Med 371, 1418–1425. [DOI] [PubMed] [Google Scholar]
  4. Barrette RW, Metwally SA, Rowland JM, Xu L, Zaki SR, Nichol ST, Rollin PE, Towner JS, Shieh WJ, Batten B, Sealy TK, Carrillo C, Moran KE, Bracht AJ, Mayr GA, Sirios-Cruz M, Catbagan DP, Lautner EA, Ksiazek TG, White WR, McIntosh MT, 2009. Discovery of swine as a host for the Reston Ebolavirus. Science 325, 204–206. [DOI] [PubMed] [Google Scholar]
  5. Biedenkopf N, Hartlieb B, Hoenen T, Becker S, 2013. Phosphorylation of Ebola virus VP30 influences the composition of the viral nucleocapsid complex: impact on viral transcription and replication. J. Biol. Chem 288, 11165–11174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brauburger K, Boehmann Y, Krähling V, Mühlberger E, 2015. Transcriptional regulation in Ebola virus: effects of gene border structure and regulatory elements on gene expression and polymerase scanning behavior. J. Virol 90, 1898–1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carroll MW, Matthews DA, Hiscox JA, Elmore MJ, Pollakis G, Rambaut A, Hewson R, Garcia-Dorival I, Bore JA, Koundouno R, Abdellati S, Afrough B, Aiyepada J, Akhilomen P, Asogun D, Atkinson B, Badusche M, Bah A, Bate S, Baumann J, Becker D, Becker-Ziaja B, Bocquin A, Borremans B, Bosworth A, Boettcher JP, Cannas A, Carletti F, Castilletti C, Clark S, Colavita F, Diederich S, Donatus A, Duraffour S, Ehichioya D, Ellerbrok H, Fernandez-Garcia MD, Fizet A, Fleischmann E, Gryseels S, Hermelink A, Hinzmann J, Hopf-Guevara U, Ighodalo Y, Jameson L, Kelterbaum A, Kis Z, Kloth S, Kohl C, Korva M, Kraus A, Kuisma E, Kurth A, Liedigk B, Logue CH, Ludtke A, Maes P, McCowen J, Mely S, Mertens M, Meschi S, Meyer B, Michel J, Molkenthin P, Munoz-Fontela C, Muth D, Newman EN, Ngabo D, Oestereich L, Okosun J, Olokor T, Omiunu R, Omomoh E, Pallasch E, Palyi B, Portmann J, Pottage T, Pratt C, Priesnitz S, Quartu S, Rappe J, Repits J, Richter M, Rudolf M, Sachse A, Schmidt KM, Schudt G, Strecker T, Thom R, Thomas S, Tobin E, Tolley H, Trautner J, Vermoesen T, Vitoriano I, Wagner M, Wolff S, Yue C, Capobianchi MR, Kretschmer B, Hall Y, Kenny JG, Rickett NY, Dudas G, Coltart CE, Kerber R, Steer D, Wright C, Senyah F, Keita S, Drury P, Diallo B, de Clerck H, Van Herp M, Sprecher A, Traore, Diakite M, Konde MK, Koivogui L, Magassouba N, Avsic-Zupanc T, Nitsche A, Strasser M, Ippolito G, Becker S, Stoecker K, Gabriel M, Raoul H, Di Caro A, Wolfel R, Formenty P, Gunther S, 2015. Temporal and spatial analysis of the 2014–2015 Ebola virus outbreak in West Africa. Nature 524, 97–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Centers for Disease Control and Prevention, 2015. Update: Ebola virus disease epidemic - West Africa, February 2015. Morb. Mortal. Wkly. Rep 64, pp. 186–187. [PMC free article] [PubMed] [Google Scholar]
  9. Centers for Disease Control and Prevention, 2016. 2014 Ebola outbreak in West Africa - case counts (March 24, 2016). Available at (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html).
  10. Chevalier MS, Chung W, Smith J, Weil LM, Hughes SM, Joyner SN, Hall E, Srinath D, Ritch J, Thathiah P, Threadgill H, Cervantes D, Lakey DL, 2014. Ebola virus disease cluster in the United States - Dallas County, Texas, 2014. Morb. Mortal. Wkly. Rep 63, pp. 1087–1088. [PMC free article] [PubMed] [Google Scholar]
  11. Dunham EC, Banadyga L, Groseth A, Chiramel AI, Best SM, Ebihara H, Feldmann H, Hoenen T, 2015. Assessing the contribution of interferon antagonism to the virulence of West African Ebola viruses. Nat. Commun 6, 8000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feldmann H, Sanchez A, Geisbert TW, 2013. Filoviridae: Marburg and Ebola viruses In: Knipe, Howley PM (Eds.), Fields Virology, 6th ed. Lippincott, Williams and Wilkins, Philadelphia, pp. 923–956. [Google Scholar]
  13. Gatherer D, 2014. The 2014 Ebola virus disease outbreak in West Africa. J. Gen. Virol 95, 1619–1624. [DOI] [PubMed] [Google Scholar]
  14. Gire SK, Goba A, Andersen KG, Sealfon RS, Park DJ, Kanneh L, Jalloh S, Momoh M, Fullah M, Dudas G, Wohl S, Moses LM, Yozwiak NL, Winnicki S, Matranga CB, Malboeuf CM, Qu J, Gladden AD, Schaffner SF, Yang X, Jiang PP, Nekoui M, Colubri A, Coomber MR, Fonnie M, Moigboi A, Gbakie M, Kamara FK, Tucker V, Konuwa E, Saffa S, Sellu J, Jalloh AA, Kovoma A, Koninga J, Mustapha I, Kargbo K, Foday M, Yillah M, Kanneh F, Robert W, Massally JL, Chapman SB, Bochicchio J, Murphy C, Nusbaum C, Young S, Birren BW, Grant DS, Scheiffelin JS, Lander ES, Happi C, Gevao SM, Gnirke A, Rambaut A, Garry RF, Khan SH, Sabeti PC, 2014. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science 345, 1369–1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartlieb B, Muziol T, Weissenhorn W, Becker S, 2007. Crystal structure of the C-terminal domain of Ebola virus VP30 reveals a role in transcription and nucleocapsid association. Proc. Natl. Acad. Sci. USA 104, 624–629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoenen T, Safronetz D, Groseth A, Wollenberg KR, Koita OA, Diarra B, Fall IS, Haidara FC, Diallo F, Sanogo M, Sarro YS, Kone A, Togo AC, Traore A, Kodio M, Dosseh A, Rosenke K, de Wit E, Feldmann F, Ebihara H, Munster VJ, Zoon KC, Feldmann H, Sow S, 2015. Mutation rate and genotype variation of Ebola virus from Mali case sequences. Science 348, 117–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG, 2000. A DNA transfection system for generation of influenza A virus eight plasmids. Proc. Natl. Acad. Sci. U S A 97, 6108–6113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacome R, Becerra A, Ponce de Leon S, Lazcano A, 2015. Structural analysis of monomeric RNA-dependent polymerases: evolutionary and therapeutic implications. PLoS One 10, e0139001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jahrling PB, Geisbert TW, Dalgard DW, Johnson ED, Ksiazek TG, Hall WC, Peters CJ, 1990. Preliminary report: isolation of Ebola virus from monkeys imported to USA. Lancet 335, 502–505. [DOI] [PubMed] [Google Scholar]
  20. Kugelman JR, Sanchez-Lockhart M, Andersen KG, Gire S, Park DJ, Sealfon R, Lin AE, Wohl S, Sabeti PC, Kuhn JH, Palacios GF, 2015a. Evaluation of the potential impact of Ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics. MBio 6, e02227–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kugelman JR, Wiley MR, Mate S, Ladner JT, Beitzel B, Fakoli L, Taweh F, Prieto K, Diclaro JW, Minogue T, Schoepp RJ, Schaecher KE, Pettitt J, Bateman S, Fair J, Kuhn JH, Hensley L, Park DJ, Sabeti PC, Sanchez-Lockhart M, Bolay FK, Palacios G, 2015b. Monitoring of Ebola virus Makona evolution through establishment of advanced genomic capability in Liberia. Emerg. Infect. Dis 21, 1135–1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuhn JH, Andersen KG, Baize S, Bao Y, Bavari S, Berthet N, Blinkova O, Brister JR, Clawson AN, Fair J, Gabriel M, Garry RF, Gire SK, Goba A, Gonzalez JP, Gunther S, Happi CT, Jahrling PB, Kapetshi J, Kobinger G, Kugelman JR, Leroy EM, Maganga GD, Mbala PK, Moses LM, Muyembe-Tamfum JJ, N’Faly M, Nichol ST, Omilabu SA, Palacios G, Park DJ, Paweska JT, Radoshitzky SR, Rossi CA, Sabeti PC, Schieffelin JS, Schoepp RJ, Sealfon R, Swanepoel R, Towner JS, Wada J, Wauquier N, Yozwiak NL, Formenty P, 2014. Nomenclature- and database-compatible names for the two Ebola virus variants that emerged in Guinea and the Democratic Republic of the Congo in 2014. Viruses 6, pp. 4760–4799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ladner JT, Wiley MR, Mate S, Dudas G, Prieto K, Lovett S, Nagle ER, Beitzel B, Gilbert ML, Fakoli L, Diclaro JW 2nd, Schoepp RJ, Fair J, Kuhn JH, Hensley LE, Park DJ, Sabeti PC, Rambaut A, Sanchez-Lockhart M, Bolay FK, Kugelman JR, Palacios G, 2015. Evolution and spread of Ebola virus in Liberia, 2014–2015. Cell Host Microbe 18, 659–669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leroy EM, Gonzalez JP, Baize S, 2011. Ebola and Marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for Africa. Clin. Microbiol. Infect 17, 964–976. [DOI] [PubMed] [Google Scholar]
  25. Liddell AM, Davey RT Jr., Mehta AK, Varkey JB, Kraft CS, Tseggay GK, Badidi O, Faust AC, Brown KV, Suffredini AF, Barrett K, Wolcott MJ, Marconi VC, Lyon GM 3rd, Weinstein GL, Weinmeister K, Sutton S, Hazbun M, Albariño CG, Reed Z, Cannon D, Stroher U, Feldman M, Ribner BS, Lane HC, Fauci AS, Uyeki TM, 2015. Characteristics and clinical management of a cluster of 3 patients with Ebola virus disease, including the first domestically acquired cases in the United States. Ann. Intern Med 163, 81–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lyon GM, Mehta AK, Varkey JB, Brantly K, Plyler L, McElroy AK, Kraft CS, Towner JS, Spiropoulou C, Stroher U, Uyeki TM, Ribner BS, 2014. Clinical care of two patients with Ebola virus disease in the United States. N Engl. J. Med 371, 2402–2409. [DOI] [PubMed] [Google Scholar]
  27. Martinez MJ, Biedenkopf N, Volchkova V, Hartlieb B, Alazard-Dany N, Reynard O, Becker S, Volchkov V, 2008. Role of Ebola virus VP30 in transcription reinitiation. J. Virol 82, 12569–12573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martinez MJ, Volchkova VA, Raoul H, Alazard-Dany N, Reynard O, Volchkov VE, 2011. Role of VP30 phosphorylation in the Ebola virus replication cycle. J. Infect. Dis 204, S934–S940. [DOI] [PubMed] [Google Scholar]
  29. Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Fulhorst CF, Rollin PE, Calaor AB, Manalo DL, Roces MC, Dayrit MM, Peters CJ, 1999. Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996. J. Infect. Dis 179, S115–S119. [DOI] [PubMed] [Google Scholar]
  30. Mühlberger E, 2007. Filovirus replication and transcription. Future Virol. 2, 205–215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Park DJ, Dudas G, Wohl S, Goba A, Whitmer SL, Andersen KG, Sealfon RS, Ladner JT, Kugelman JR, Matranga CB, Winnicki SM, Qu J, Gire SK, Gladden-Young A, Jalloh S, Nosamiefan D, Yozwiak NL, Moses LM,Jiang PP, Lin AE, Schaffner SF, Bird B, Towner J, Mamoh M, Gbakie M, Kanneh L, Kargbo D, Massally JL, Kamara FK, Konuwa E, Sellu J, Jalloh AA, Mustapha I, Foday M, Yillah M, Erickson BR, Sealy T, Blau D, Paddock C, Brault A, Amman B, Basile J, Bearden S, Belser J, Bergeron E, Campbell S, Chakrabarti A, Dodd K, Flint M, Gibbons A, Goodman C, Klena J, McMullan L, Morgan L, Russell B, Salzer J, Sanchez A, Wang D, Jungreis I, Tomkins-Tinch C, Kislyuk A, Lin MF, Chapman S, MacInnis B, Matthews A, Bochicchio J, Hensley LE, Kuhn JH, Nusbaum C, Schieffelin JS, Birren BW, Forget M, Nichol ST, Palacios GF, Ndiaye D, Happi C, Gevao SM, Vandi MA, Kargbo B, Holmes EC, Bedford T, Gnirke A, Ströher U, Rambaut A, Garry RF, Sabeti PC, 2015. Ebola virus epidemiology, transmission, and evolution during seven months in Sierra Leone. Cell 161, 1516–1526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rollin PE, Williams RJ, Bressler DS, Pearson S, Cottingham M, Pucak G, Sanchez A, Trappier SG, Peters RL, Greer PW, Zaki S, Demarcus T, Hendricks K, Kelley M, Simpson D, Geisbert TW, Jahrling PB, Peters CJ, Ksiazek TG, 1999. Ebola (subtype Reston) virus among quarantined nonhuman primates recently imported from the Philippines to the United States. J. Infect. Dis 179, S108–S114. [DOI] [PubMed] [Google Scholar]
  33. Rougeron V, Feldmann H, Grard G, Becker S, Leroy EM, 2015. Ebola and Marburg haemorrhagic fever. J. Clin. Virol 64, 111–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tong YG, Shi WF, Liu D, Qian J, Liang L, Bo XC, Liu J, Ren HG, Fan H, Ni M, Sun Y, Jin Y, Teng Y, Li Z, Kargbo D, Dafae F, Kanu A, Chen CC, Lan ZH, Jiang H, Luo Y, Lu HJ, Zhang XG, Yang F, Hu Y, Cao YX, Deng YQ, Su HX, Sun Y, Liu WS, Wang Z, Wang CY, Bu ZY, Guo ZD, Zhang LB, Nie WM, Bai CQ, Sun CH, An XP, Xu PS, Zhang XL, Huang Y, Mi ZQ, Yu D, Yao HW, Feng Y, Xia ZP, Zheng XX, Yang ST, Lu B, Jiang JF, Kargbo B, He FC, Gao GF, Cao WC, 2015. Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone. Nature 524, 93–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Uebelhoer LS, Albariño CG, McMullan LK, Chakrabarti AK, Vincent JP, Nichol ST, Towner JS, 2014. High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses. Antivir. Res 106, 86–94. [DOI] [PubMed] [Google Scholar]
  36. Volchkov VE, Volchkova VA, Chepurnov AA, Blinov VM, Dolnik O, Netesov SV, Feldmann H, 1999. Characterization of the L gene and 5’ trailer region of Ebola virus. J. Gen. Virol 80, 355–362. [DOI] [PubMed] [Google Scholar]
  37. WHO, 2016. Ebola Situation Report −16 March 2016. Avaliable at (http://apps.who.int/ebola/current-situation/ebola-situation-report-16-march-2016).

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Supplementary Materials

Suppl.Fig.1
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Suppl.Fig.2
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Suppl.Fig.3
NIHMS917928-supplement-Suppl_Fig_3.tiff (425.1KB, tiff)

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