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PostPosted: Thu Aug 28, 2014 2:58 pm 

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Science paper describes 99 Ebola sequences from Sierra Leone cases

http://www.sciencemag.org/content/early ... 59657.full


PostPosted: Thu Aug 28, 2014 2:59 pm 

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Published Online August 28 2014
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Science DOI: 10.1126/science.1259657 •Report

Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak

Stephen K. Gire1,2,*,
Augustine Goba3,*,†,
Kristian G. Andersen1,2,*,†,
Rachel S. G. Sealfon2,4,*,
Daniel J. Park2,*,
Lansana Kanneh3,
Simbirie Jalloh3,
Mambu Momoh3,5,
Mohamed Fullah3,5,‡,
Gytis Dudas6,
Shirlee Wohl1,2,7,
Lina M. Moses8,
Nathan L. Yozwiak1,2,
Sarah Winnicki1,2,
Christian B. Matranga2,
Christine M. Malboeuf2,
James Qu2,
Adrianne D. Gladden2,
Stephen F. Schaffner1,2,
Xiao Yang2,
Pan-Pan Jiang1,2,
Mahan Nekoui1,2,
Andres Colubri1,
Moinya Ruth Coomber3,
Mbalu Fonnie3,‡,
Alex Moigboi3,‡,
Michael Gbakie3,
Fatima K. Kamara3,
Veronica Tucker3,
Edwin Konuwa3,
Sidiki Saffa3,
Josephine Sellu3,
Abdul Azziz Jalloh3,
Alice Kovoma3,‡,
James Koninga3,
Ibrahim Mustapha3,
Kandeh Kargbo3,
Momoh Foday3,
Mohamed Yillah3,
Franklyn Kanneh3,
Willie Robert3,
James L. B. Massally3,
Sinéad B. Chapman2,
James Bochicchio2,
Cheryl Murphy2,
Chad Nusbaum2,
Sarah Young2,
Bruce W. Birren2,
Donald S. Grant3,
John S. Scheiffelin8,
Eric S. Lander2,7,9,
Christian Happi10,
Sahr M. Gevao11,
Andreas Gnirke2,§,
Andrew Rambaut6,12,13,§,
Robert F. Garry8,§,
S. Humarr Khan3,‡§,
Pardis C. Sabeti1,2,†§

+ Author Affiliations

1Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.

2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

3Kenema Government Hospital, Kenema, Sierra Leone.

4Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

5Eastern Polytechnic College, Kenema, Sierra Leone.

6Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 3JT, UK.

7Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

8Tulane University Medical Center, New Orleans, LA 70112, USA.

9Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

10Redeemer’s University, Ogun State, Nigeria.

11University of Sierra Leone, Freetown, Sierra Leone.

12Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA.

13Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh EH9 3JT, UK.

+ Author Notes

↵‡ Deceased.

↵†Corresponding author. E-mail: andersen@broadinstitute.org (K.G.A.); augstgoba@yahoo.com (A.G.); psabeti@oeb.harvard.edu (P.C.S.)

↵* These authors contributed equally to this work.

↵§ These authors jointly supervised this work.


PostPosted: Thu Aug 28, 2014 3:16 pm 

Joined: Wed Aug 19, 2009 10:42 am
Posts: 56044
Location: Pittsburgh, PA USA
In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2,000x coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic. This West African variant likely diverged from Middle African lineages ~2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources. Since many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.


PostPosted: Thu Aug 28, 2014 3:17 pm 

Joined: Wed Aug 19, 2009 10:42 am
Posts: 56044
Location: Pittsburgh, PA USA
Ebola virus (EBOV; formerly Zaire ebolavirus), one of five ebolaviruses, is a lethal human pathogen, causing Ebola virus disease (EVD) with an average case fatality rate of 78% (1). Previous EVD outbreaks were confined to remote regions of Middle Africa; the largest, in 1976, had 318 cases (2) (Fig. 1A). The current outbreak started in February 2014 in Guinea, West Africa (3) and spread into Liberia in March, Sierra Leone in May, and Nigeria in late July. It is the largest known EVD outbreak and is expanding exponentially with a doubling period of 34.8 days (Fig. 1B). As of August 19th, 2,240 cases and 1,229 deaths have been documented (4, 5). Its emergence in the major cities of Conakry (Guinea), Freetown (Sierra Leone), Monrovia (Liberia), and Lagos (Nigeria) raises the specter of increasing local and international dissemination.

Fig. 1
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Fig. 1 Ebola outbreaks, historical and current.
(A) Historical EVD outbreaks, colored by decade. Circle area represents total number of cases (RC = Republic of the Congo; DRC = Democratic Republic of Congo). (B) 2014 outbreak growth (confirmed, probable and suspected cases). (C) Spread of EVD in Sierra Leone by district. The gradient denotes number of cases and the arrows depict likely direction. (D) EBOV samples from 78 patients were sequenced in two batches, totaling 99 viral genomes (Replication = technical replicates (6)). Mean coverage and median depth of coverage with range are shown. (E) Combined normalized (to the sample average) coverage across sequenced EBOV genomes.

In an ongoing public health crisis, where accurate and timely information is crucial, new genomic technologies can provide near real-time insights into the pathogen’s origin, transmission dynamics, and evolution. We used massively parallel viral sequencing to understand how and when EBOV entered human populations in the 2014 West African outbreak, whether the outbreak is continuing to be fed by new transmissions from its natural reservoir, and how the virus changed, both before and after its recent jump to humans.

In March 2014, Kenema Government Hospital (KGH) established EBOV surveillance in Kenema, Sierra Leone, near the origin of the 2014 outbreak (Fig. 1C and fig. S1) (6). Following standards for field-based tests in previous (7) and current (3) outbreaks, KGH performed conventional PCR-based EBOV diagnostics (8) (fig. S2); all tests were negative through early May. On May 25, KGH scientists confirmed the first case of EVD in Sierra Leone. Investigation by the Ministry of Health and Sanitation (MoHS) uncovered an epidemiological link between this case and the burial of a traditional healer who had treated EVD patients in Guinea. Tracing led to 13 additional cases—all females who attended the burial. We obtained ethical approval from MoHS, the Sierra Leone Ethics and Scientific Review Committee, and our U.S. institutions to sequence patient samples in the U.S. using approved safety standards (6).

We evaluated four independent library preparation methods and two sequencing platforms (9) (table S1) for our first batch of 15 inactivated EVD samples from 12 patients. Nextera library construction and Illumina sequencing provided the most complete genome assembly and reliable intrahost single nucleotide variant (iSNV, frequency >0.5%) identification (6). We used this combination for a second batch of 84 samples from 66 additional patients, performing two independent replicates from each sample (Fig. 1D). We also sequenced 35 samples from suspected EVD cases that tested negative for EBOV; genomic analysis identified other known pathogens, including Lassa virus, HIV-1, enterovirus A and malaria parasites (fig. S3).

In total, we generated 99 EBOV genome sequences from 78 confirmed EVD patients, representing over 70% of the EVD patients diagnosed in Sierra Leone in late May to mid June; we employed multiple extraction methods or timepoints for 13 patients (table S2). Median coverage was >2,000x, spanning more than 99.9% of EBOV coding regions (Fig. 1, D and E, and table S2).

We combined the 78 Sierra Leonean sequences with 3 published Guinean samples (3) (correcting 21 likely sequencing errors in the latter (6)) to obtain a dataset of 81 sequences. They reveal 341 fixed substitutions between the 2014 EBOV and all previously published EBOV (35 nonsynonymous, 173 synonymous, 133 noncoding), with an additional 55 single nucleotide polymorphisms (SNPs) (fixed within individual patients) within the West African outbreak (15 nonsynonymous, 25 synonymous, 15 noncoding). Notably, the Sierra Leonean genomes differ from PCR probes for five separate assays used for EBOV and pan-filovirus diagnostics (table S3).

Deep-sequence coverage allowed identification of 263 iSNVs (73 nonsynonymous, 108 synonymous, 70 noncoding, and 12 frameshift) in the Sierra Leone patients (6). For all patients with multiple time points, consensus sequences were identical and iSNV frequencies remained stable (fig. S4). One notable intrahost variation is the RNA editing site of the glycoprotein (GP) gene (fig. S5A) (10–12), which we characterize in patients (6).

Phylogenetic comparison to all 20 genomes from earlier outbreaks suggests the 2014 West African virus likely spread from Middle Africa within the last decade. Rooting the phylogeny using divergence to other ebolavirus genomes is problematic (Fig. 2A and fig. S6) (6, 13). However, rooting the tree on the oldest outbreak reveals a strong correlation between sample date and root-to-tip distance, with a substitution rate of 8x10−4/site/year (Fig. 2B and fig. S7) (13). This suggests that the lineages of the three most recent outbreaks all diverged from a common ancestor at roughly the same time c. 2004 (Fig. 2C and Fig. 3A), supporting the hypothesis that each outbreak represents an independent zoonotic event from the same genetically diverse viral population in its natural reservoir.

Fig. 2
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Fig. 2 Relationship between outbreaks.
(A) Unrooted phylogenetic tree of EBOV samples; each major clade corresponds to a distinct outbreak (scale bar = nucleotide substitutions/site). (B) Root-to-tip distance correlates better with sample date when rooting on the 1976 branch (R2 = 0.92, top) than on the 2014 branch (R2 = 0.67, bottom). (C) Temporally rooted tree from (A).

Fig. 3
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Fig. 3 Molecular dating of the 2014 outbreak.
(A) BEAST dating of the separation of the 2014 lineage from Middle African lineages (SL = Sierra Leone; GN = Guinea; DRC = Democratic Republic of Congo; tMRCA: Sep 2004, 95% HPD: Oct 2002 - May 2006). (B) BEAST dating of the tMRCA of the 2014 West African outbreak (tMRCA: Feb 23, 95% HPD: Jan 27 - Mar 14) and the tMRCA of the Sierra Leone lineages (tMRCA: Apr 23, 95% HPD: Apr 2 - May 13); probability distributions for both 2014 divergence events overlayed below. Posterior support for major nodes is shown.

Genetic similarity across the sequenced 2014 samples suggests a single transmission from the natural reservoir, followed by human-to-human transmission during the outbreak. Molecular dating places the common ancestor of all sequenced Guinea and Sierra Leone lineages around late February 2014 (Fig. 3B), three months after the earliest suspected cases in Guinea (3); this coalescence would be unlikely had there been multiple transmissions from the natural reservoir. Thus, in contrast to some previous EVD outbreaks (14), continued human-reservoir exposure is unlikely to have contributed to the growth of this epidemic in areas represented by available sequence data.

Our data suggest the Sierra Leone outbreak stemmed from the introduction of two genetically distinct viruses from Guinea around the same time. Samples from 12 of the first EVD patients in Sierra Leone, all believed to have attended the funeral of an EVD case from Guinea, fall into two distinct clusters (clusters 1 and 2) (Fig. 4A and fig. S8). Molecular dating places the divergence of these two lineages in late April (Fig. 3B), predating their co-appearance in Sierra Leone in late May (Fig. 4B), suggesting the funeral attendees were most likely infected by two lineages then circulating in Guinea, possibly at the funeral (fig. S9). All subsequent diversity in Sierra Leone accumulated on the background of those two lineages (Fig. 4A), consistent with epidemiological information from tracing contacts.

Fig. 4
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Fig. 4 Viral dynamics during the 2014 outbreak.
(A) Mutations, one patient sample per row; beige = identical to Kissidougou Guinean sequence (accession KJ660346). The top row shows the type of mutation (green: synonymous, pink: nonsynonymous, intergenic: gray), with genomic locations indicated above. Clusters assignments are shown on left. (B) Number of EVD-confirmed patients per day, colored by cluster (arrow: first appearance of the derived allele at position 10,218, distinguishing clusters 2 and 3). (C) Intrahost frequency of SNP 10,218 in all 78 patients (absent in 28 patients, polymorphic in 12, fixed in 38). (D and E) 12 patients carrying iSNV 10,218 cluster geographically and temporally (HCW-A = unsequenced health care worker, Driver drove HCW-A from Kissi Teng to Jawie, then continued alone to Mambolo, HCW-B treated HCW-A). (F) Substitution rates within the 2014 outbreak and between all EVD outbreaks. (G) Proportion of nonsynonymous changes observed on different time scales (green = synonymous; pink = nonsynonymous). (H) Acquisition of genetic variation over time. 50 mutational events (short dashes) and 29 new viral lineages (long dashes) were observed (intrahost variants not included).

Patterns in observed intrahost and interhost variation provide important insights about transmission and epidemiology. Groups of patients with identical viruses or with shared intrahost variation show temporal patterns suggesting transmission links (fig. S10). One iSNV (position 10,218) shared by twelve patients is later observed as fixed within 38 patients, becoming the majority allele in the population (Fig. 4C) and defining a third Sierra Leone cluster (Fig. 4, A and D, and fig. S8). Repeated propagation at intermediate frequency suggests that transmission of multiple viral haplotypes may be common. Geographic, temporal, and epidemiological metadata supports the transmission clustering inferred from genetic data (Fig. 4, D and E, and fig. S11) (6).

The observed substitution rate is roughly twice as high within the 2014 outbreak as between outbreaks (Fig. 4F). Mutations are also more frequently nonsynonymous during the outbreak (Fig. 4G). Similar findings have been seen previously (15) and are consistent with expectations from incomplete purifying selection (16–18). Determining whether individual mutations are deleterious, or even adaptive, would require functional analysis; however, the rate of nonsynonymous mutations suggests that continued progression of this epidemic could afford an opportunity for viral adaptation (Fig. 4H), underscoring the need for rapid containment.

As in every EVD outbreak, the 2014 EBOV variant carries a number of genetic changes distinct to this lineage; our data do not address whether these differences are related to the severity of the outbreak. However, the catalog of 395 mutations, including 50 fixed nonsynonymous changes with 8 at positions with high levels of conservation across ebolaviruses, provide a starting point for such studies (table S4).

To aid in relief efforts and facilitate rapid global research, we immediately released all sequence data as generated. Ongoing epidemiological and genomic surveillance is imperative to identify viral determinants of transmission dynamics, monitor viral changes and adaptation, ensure accurate diagnosis, guide research on therapeutic targets, and refine public-health strategies. It is our hope that this work will aid the multidisciplinary, international efforts to understand and contain this expanding epidemic.

In memoriam: Tragically, five co-authors, who contributed greatly to public health and research efforts in Sierra Leone, contracted EVD in the course of their work and lost their battle with the disease before this manuscript could be published. We wish to honor their memory.

Supplementary Materials

Materials and Methods
Supplementary Text
Figs. S1 to S11
Tables S1 to S4
Supplementary files S1 to S4
References (19–44)
Received for publication 5 August 2014.
Accepted for publication 21 August 2014.


PostPosted: Thu Aug 28, 2014 3:18 pm 

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Acknowledgments: We thank the Sierra Leone MoHS (Hon. Minister M. Kargbo, B. Kargbo, M.A. Vandi, A. Jambai), the Kenema District Health Management Team and Lassa fever program for their efforts in outbreak response. We thank P. Cingolani, Y.-C. Wu, M. Lipsitch, S. Günther, S. Baize, N. Wauquier, J. Bangura, V. Lungay, L. Hensley, J. Johnson, M. Voorhees, A. O’Hearn, and R. Schoepp, L. Gaffney, J. Kuhn, S.C. Sealfon, J.B. Shapiro, C. Edwards, Sabeti lab members for technical support and feedback. This project has been funded in part by NIH 1DP2OD006514-01 and NIAID HHSN272200900049C. RS is supported by NSF GRFP, SW by NIH GM080177; CH by NIH 1U01HG007480-01 and the World Bank; AR by EU FP7/2007-2013 278433-PREDEMICS and ERC 260864; and GD by NERC D76739X. Sequence data are available at NCBI (NCBI BioGroup: PRJNA257197). Sharing of RNA samples used in this study requires approval from the Sierra Leone Ministry of Health and Sanitation.


PostPosted: Thu Aug 28, 2014 3:31 pm 

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Science 29 August 2014:
Vol. 345 no. 6200 pp. 989-990
DOI: 10.1126/science.345.6200.989
•In Depth

Infectious Disease

Genomes reveal start of Ebola outbreak

Gretchen Vogel



PostPosted: Thu Aug 28, 2014 3:32 pm 

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Location: Pittsburgh, PA USA
When the young woman arrived at the Kenema Government Hospital in Sierra Leone in late May, she had high fever and had just miscarried. The hospital suspected she had contracted Lassa fever, because the viral disease is endemic in the region and often causes miscarriages. But Ebola virus disease, another hemorrhagic fever illness, had been spreading in neighboring Guinea for months, so when she began bleeding profusely, staff tested her for that virus as well. The results were positive, making her the first confirmed case of Ebola in Sierra Leone.

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Volunteers in protective gear bury a victim of Ebola in Kenema.


The young woman, who eventually recovered, is now at the heart of a tragic but potentially important research tale. In a paper online this week in Science, a collaboration led by Stephen Gire and Pardis Sabeti of Harvard University and the Broad Institute in Cambridge, Massachusetts, report sequencing and analyzing the genomes of Ebola virus samples from 78 people in Sierra Leone who were diagnosed with Ebola between late May and mid-June, including the young woman who came to Kenema's hospital. The 99 complete sequences—some patients were sampled more than once—provide insights into how the virus is changing during the outbreak, which could help improve current diagnostic tests and, in the long term, guide researchers working on vaccines and treatments.

The study, however, also highlights the unrelenting toll the outbreak has taken on health workers on the front lines. More than 50 co-authors from four countries helped collect and analyze the viral sequences. Five of them contracted Ebola virus disease themselves and died.

That first diagnosed case in Sierra Leone infected no one at the hospital, says Robert Garry, a virologist at Tulane University in New Orleans, Louisiana, who works with the Kenema hospital's Lassa fever research center and is also a co-author on the paper. But a team from the ministry of health was immediately dispatched to the woman's home village to find out where and how she had been infected. They learned that she had attended the recent funeral of a traditional healer—an herbalist—who had been treating Ebola patients from across the nearby border with Guinea.

The team found 13 more people who were infected, all women who had attended the burial. It was those mourners who largely sparked Sierra Leone's outbreak, which has sickened more than 900 and killed more than 390 people. Blood samples from 12 of those mourners and other infected people have allowed Gire, Sabeti, and their colleagues to track how the virus changed as it spread. “It is the first time that the real evolution of the Ebola virus can be observed in humans,” says Sylvain Baize of the Institut Pasteur in Lyon, France, who sequenced some of the first Ebola virus samples from patients in Guinea, where the current outbreak originated, but who was not involved in this project.

The genomic data also shed new light on how the virus—officially called EBOV—ended up in West Africa. EBOV, one of five ebolaviruses known to infect humans, has caused at least 12 outbreaks in Central Africa and Gabon since 1976. Until this year, though, it had never been identified in West Africa.

Some researchers theorized, based on early sequencing data, that EBOV had circulated for decades, undetected, in animals in the region. But the new analysis, strengthened by the unprecedented number of genomes, supports another theory: that the virus spread, via animal hosts, from Central Africa within the last decade. Researchers aren't sure which animal to blame, but fruit bats are their leading suspects (Science, 11 April, p. 140). At least one fruit bat species known to carry ebolavirus has a population range that stretches from Central Africa across to Guinea.

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Gire, Sabeti, and their colleagues found that in the current outbreak the virus's genome is changing fairly quickly, including in regions that are key for the accuracy of the PCR-based diagnostic tests. It will be important to keep track of such changes, Gire says, so that tests can be updated if necessary. Vaccines and antibody-based treatments—such as the ZMapp drug that was used in a handful of patients—could also be affected by the kinds of changes the researchers identified. (Sabeti says ZMapp researchers contacted her about the new sequences her group had posted online.)

The analysis reveals that the outbreak in Sierra Leone was sparked by at least two distinct viruses, introduced from Guinea at about the same time. It is unclear whether the herbalist was infected with both variants, or whether perhaps another funeral attendee was independently infected. One Ebola virus lineage disappears from patient samples taken later in the outbreak, while a third lineage appears. That lineage—tied to a nurse who was traveling to reach a hospital but died along the way—seems to have originated when one of the lineages present at the funeral gained a new mutation. This third lineage was spread, Garry says, via a truck driver who transported the nurse, as well as others who cared for her in the town where she died.

Further studies of the differences between the various Ebola lineages might link such mutations to the virus's behavior—how lethal it is, and how easily it spreads, for example. “The paper shows the unrealized potential of what these methods could do,” says Roman Biek, who studies the evolution and ecology of infectious diseases at the University of Glasgow in the United Kingdom.

Missing from the sequencing analysis are Ebola samples from people infected in Liberia and Guinea. Stephan Günther of the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, says he has samples from Guinea in his lab, waiting to be sequenced once he and colleagues can find the time. (This week Günther was in Nigeria, tracing contacts of an Ebola patient there, who was infected by a traveler from Liberia.) Researchers in Liberia have also collected samples, but are focused on attempting to slow the epidemic there, where it is spreading in the densely populated capital and shows no signs of slowing down. (Congo is also on high alert as Ebola has popped up in a remote region in the northwest of the country. As Science went to press, it was not clear which ebolavirus is causing that outbreak.)

Sabeti, who with her colleagues posted the virus sequences in a public database as soon as they were generated, says she hopes this work and the tragedy that befell her co-authors and other health care workers will inspire other researchers to make their data public quickly, in both this outbreak and future epidemics. “We've got to crowdsource the epidemic,” she says. “The more information you get into hands of people who can help, the more likely you are to come up with a solution.”

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In Science Magazine


Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak
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Lansana Kanneh,
Simbirie Jalloh,
Mambu Momoh,
Mohamed Fullah,
Gytis Dudas,
Shirlee Wohl,
Lina M. Moses,
Nathan L. Yozwiak,
Sarah Winnicki,
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Christine M. Malboeuf,
James Qu,
Adrianne D. Gladden,
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Pan-Pan Jiang,
Mahan Nekoui,
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Moinya Ruth Coomber,
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Michael Gbakie,
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Edwin Konuwa,
Sidiki Saffa,
Josephine Sellu,
Abdul Azziz Jalloh,
Alice Kovoma,
James Koninga,
Ibrahim Mustapha,
Kandeh Kargbo,
Momoh Foday,
Mohamed Yillah,
Franklyn Kanneh,
Willie Robert,
James L. B. Massally,
Sinéad B. Chapman,
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Cheryl Murphy,
Chad Nusbaum,
Sarah Young,
Bruce W. Birren,
Donald S. Grant,
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Eric S. Lander,
Christian Happi,
Sahr M. Gevao,
Andreas Gnirke,
Andrew Rambaut,
Robert F. Garry,
S. Humarr Khan,
and Pardis C. Sabeti

Science 1259657Published online 28 August 2014
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PostPosted: Thu Aug 28, 2014 3:35 pm 

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Ebola Outbreak Strains Sequenced
Ninety-nine publicly available genomes could help researchers working to develop diagnostics, vaccines, and therapies.

By Tracy Vence | August 28, 2014

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Augustine Goba of Kenema Government Hospital diagnosed the first case of Ebola in Sierra Leone.

An international team led by investigators at Harvard University has sequenced 99 Ebola virus genomes isolated from the blood of 78 patients in Sierra Leone—one of four countries at the center of the largest-ever Ebola outbreak. Within these sequences, which were each made public within a matter of days post-assembly, the researchers found evidence of the rapid accumulation of mutations affecting biologically meaningful targets, which could have implications for the development of diagnostics, vaccines, and therapies. The team’s analysis of all 99 genomes was published today (August 28) in Science.

“This analysis will provide the backbone for tracking the virus as it spreads, and to see if future outbreaks outside of these countries are connected both epidemiologically and genetically,” emerging infectious diseases researcher Matthew Frieman from the University of Maryland School of Medicine who was not involved in the work told The Scientist in an e-mail. “The ability to deep sequence virus samples rapidly, inexpensively, and safely has opened up a window in to genomic surveillance that did not exist before.”

Harvard’s Pardis Sabeti and her colleagues have been working to detect signs of natural selection in the Ebola virus genome for the last five years. With the help of collaborators from Tulane University Medical Center, other US institutions, and hospitals in Sierra Leone and Nigeria, Sabeti’s team had already established biosecurity level-4 diagnostic testing infrastructure in West Africa before the first signs of an Ebola outbreak emerged in Guinea this February. In March, study coauthors Stephen Gire and Kristian Andersen traveled to Sierra Leone and, along with coauthor Augustine Goba from the country’s Kenema Government Hospital, began testing patients for Ebola.

“In May, they had our first positive case,” said Sabeti. “When that happened, we moved very quickly.”

Once cleared by the Sierra Leonean health ministry, Gire, Andersen, Goba, and their colleagues began shipping patient samples to the Broad Institute in Cambridge, Massachusetts, where Sabeti is a senior associate member. The Broad team sequenced each sample six times at about 2,000x coverage.

“This dataset allowed the authors to provide a detailed and unprecedented account of the evolution and spread of Ebola virus within Sierra Leone during a portion of the ongoing epidemic,” Jason Ladner from the US Army Medical Research Institute of Infectious Diseases in Fort Detrick, Maryland, wrote in an e-mail to The Scientist. “In particular, these sequences have provided the first look into how the virus has been changing since the start of the outbreak. Such genetic changes are important to account for in the design and testing of medical countermeasures.”

Shortly after completing the sequencing work, Sabeti and her colleagues made the results publicly available online, and almost immediately they began receiving calls from researchers working to develop diagnostics, vaccines, and therapies. “We wanted as many eyes looking at the data as quickly as possible,” said Sabeti. “Already we’ve seen that the virus has mutated away from currently used diagnostics, [which] is likely to have some effect on the sensitivity of those assays.”

Specifically, the team’s analysis suggested that the Ebola virus strain circulating in West Africa diverged from Middle African lineages around 2004, and has since shown sustained human-to-human transmission. While the researchers were unable to pinpoint the original animal source of the virus, they did not find any evidence of additional zoonotic sources in the outbreak strains.

“The organism is changing all the time and that can create problems,” said University of Warwick microbial genomicist Mark Pallen. To devise effective countermeasures, he added, “you need to be clear which parts of the genome are staying fairly static.”

“This sequence analysis lets us know where it [the virus] went and gives scientists a scaffold to look for where it came from,” added Frieman.

The World Health Organization (WHO), which earlier this month declared the ongoing epidemic a Public Health Emergency of International Concern, today (August 28) said that nearly 20,000 people could be infected with Ebola before the outbreak is contained. According to the WHO, at least 3,069 people have been infected with the virus to date; 1,552 have died.

“We’ve been really overwhelmed by what’s going on,” said Sabeti. “This is an extraordinary emergency on an unprecedented scale.”

Five healthcare workers who assisted the team on the ground in Sierra Leone died of Ebola. All five were infected with the virus while caring for sick patients or family members, Sabeti said. In their paper, the authors honored their deceased colleagues—five would-be coauthors on the work—who gave their lives to help others.

“These individuals were heroic and battling something extraordinarily dangerous,” said Sabeti. “Without them, this wouldn’t be possible.”

S.K. Gire et al., “Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak,” Science, doi: 10.1126/science.1259657, 2014.

http://www.the-scientist.com/?articles. ... Sequenced/


PostPosted: Thu Aug 28, 2014 3:40 pm 

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Ebola virus mutating rapidly as it spreads
Outbreak likely originated with a single animal-to-human transmission.

Erika Check Hayden
28 August 2014
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On 24 May, Augustine Goba received a blood sample from a pregnant woman in Sierra Leone who had fallen ill after attending the funeral of an Ebola victim in Guinea. Twenty-four hours later, the test results came back positive. Goba, who directs a diagnostic lab at Kenema Government Hospital in Sierra Leone, had confirmed the country's first case of Ebola.

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He and his colleagues have now decoded the genetic sequences of 99 Ebola viruses collected from 78 patients during the first 24 days of the epidemic in Sierra Leone. The work1, published online in Science, could help to inform the design of diagnostics, therapeutics and vaccines, says structural biologist Erica Ollmann Saphire of The Scripps Research Institute in La Jolla, California. “This paper is terrific,” she adds.

The Ebola epidemic in West Africa has already killed more than 1,400 people — including five of Goba's co-authors from Kenema. The paper is dedicated to their memory.

The sequence data, which were made publicly available by 31 July, constitute the largest collection of genetic information on Ebola ever to be released. To get them, the group collected leftover blood from samples taken for diagnostic tests in Kenema. They then used a chemical solution to deactivate the Ebola viruses, and sent the samples to be sequenced at the Broad Institute in Cambridge, Massachusetts.

The researchers sequenced the viral genomes from each sample an average of more than 2,000 times, allowing them track how the virus mutated as it spread from patient to patient. In April, researchers reported2 that they had sequenced data from Guinean patients' viruses. That team, however, produced one composite viral genome sequence for each patient, rather than individually sequencing different copies of the virus found in each patient, as in the work reported today.

Stephen Gire
Augustine Goba confirmed the first case of Ebola virus in Sierra Leone.
Back to the beginning
By comparing their data to the Guinean sequence data, Goba's team confirmed that Ebola was probably imported to Sierra Leone by 12 people who attended the funeral in Guinea, and that the West African outbreak originated in a single event in which the virus passed from an animal into a person. Further comparisons suggest that the virus that caused the outbreak separated from those that caused past Ebola outbreaks about 10 years ago. It had accumulated more than 395 mutations between that time and June, when the researchers collected the last samples included in today's analysis.

The virus amassed 50 mutations during its first month, the researchers found. The researchers say there is no sign that any of these mutations have contributed to the unprecedented size of the outbreak by changing the characteristics of the Ebola virus - for instance, its ability to spread from person to person or to kill infected patients. But others are eager to examine these questions.

And such risks rise as the virus continues to spread. “The longer we allow the outbreak to continue, the greater the opportunity the virus has to mutate, and it’s possible that it will mutate into a form that would be an even greater threat than it is right now,” says Charles Chiu, an infectious-disease physician at the University of California, San Francisco.

Constant monitoring
The mutations do not seem to be affecting the efficacy of experimental drugs and vaccines, some of which have been given to patients in this outbreak. Some changes have occurred in regions of the genome that are targeted by diagnostic tests. This does not mean the tests are ineffective, but confirming this and continuing to monitor such mutations will be crucial, Chiu says.

Related stories
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Should experimental drugs be used in the Ebola outbreak?
Ebola declared a public-health emergency
More related stories
In the meantime, doctors and researchers say that the only way to end the outbreak is to send more health workers and supplies to affected regions, and to train Africans to diagnose, trace and treat Ebola.

Several authors of the study, including Christian Happi of Redeemer’s University in Redemption City, Nigeria, have been involved in such training in West Africa, and are now preparing researchers there to perform genetic sequencing. Happi’s African Centre of Excellence for Genomics of Infectious Disease at the university is expecting to receive the first next-generation sequencer in West Africa.

“Our hope is that next time this happens, we will be able to perform deep sequencing right on African soil,” Happi says.

Nature doi:10.1038/nature.2014.15777
Follow Erika on Twitter @Erika_Check.

Gire, S. K. et al. Science http://www.sciencemag.org/lookup/doi/10 ... ce.1259657 (2014).
Show context
Baize, S. et al. N. Engl. J. Med. http://dx.doi.org/10.1056/NEJMoa1404505 (2014).
Show context

http://www.nature.com/news/ebola-virus- ... NatureNews


PostPosted: Thu Aug 28, 2014 3:46 pm 

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Location: Pittsburgh, PA USA
Scientists found the origins of the Ebola outbreak — by tracking its mutations
Updated by Susannah Locke on August 28, 2014, 2:00 p.m. ET @susannahlocke susannah@vox.com


Sierra Leone government burial team members wearing protective clothing carry the coffin of Dr Modupeh Cole, Sierra Leone's second senior physician to die of Ebola. Carl De Souza/AFP/Getty Images
One of the big mysteries in the Ebola outbreak in West Africa is where the virus came from in the first place — and whether it's changed in any significant ways. These unanswered questions could be making it more difficult to diagnose the disease and find treatments.


Now scientists are starting to get some answers. In a new paper in Science, researchers reveal that they have sequenced the genomes of Ebola from 78 patients in Sierra Leone who contracted the disease in May and June. Those sequences revealed some 300 mutations specific to this outbreak.

The new analysis could help determine if the virus' behavior has changed — and provide information for future diagnostic tests and treatments.

Among their findings, the researchers discovered that the current viral strains come from a related strain that left Central Africa within the past ten years. And the research confirms that the virus likely spread into Sierra Leone when women became infected after attending the funeral of a traditional healer who had been treating Guinean Ebola patients.

The current Ebola outbreak in West Africa is the worst on record. It has hit four countries, including Sierra Leone, infected approximately 3,000, and killed about 1,500 people. And so far, there is no sign of it slowing down.

The fact that the researchers were able to sequence the Ebola genomes in mere months is remarkable — a contrast to the typically slow pace of scientific research. "We’re trying to do this as fast as possible," says co-senior author Pardis Sabeti, a biologist at MIT and Harvard. This new data increases the number of public Ebola virus sequences fourfold.

The main impact of the paper will be as the foundation of research for years to come as other projects try to sort out what all of these genetic sequences — and their hundreds of mutations — really mean.

The paper is also a sad reminder of the toll that the virus has taken on those working on the front lines. Five of the authors died of Ebola before it was published. All were affiliated with Kenema Government Hospital in Sierra Leone.

What genetic sequences can tell us about Ebola

Ebola tree lineage
(Gire, SK, et al. Science, August 28, 2014.)

Viruses randomly mutate over time. This is completely normal for viruses, and there's no reason to think that Ebola's mutation rate is anything weird or unusual.


Scientists can use these mutations as markers to piece together how the Ebola virus has traveled from person to person. Because they know the general mutation rate of the virus, they can also pin down the dates of when the disease spread.

So what has this analysis revealed? Using genetic sequences from current and previous outbreaks, the researchers mapped out a family tree that puts a common ancestor of the recent West African outbreak someplace in Central Africa roughly around 2004. This contradicts an earlier hypothesis that the virus had been hanging around West Africa for much longer than that.

The data, on the whole, supports what epidemiologists have already deduced about how the virus spread into Sierra Leone. More than a dozen women became infected after attending the funeral of a traditional healer who had been treating Guinean Ebola patients and contracted the disease.

One surprise from the paper is that two different strains of Ebola came out of that funeral. This suggests that either the healer was infected with two different strains or that another person at the funeral was already infected.

As Ebola then traveled across Sierra Leone, a third strain of the virus appeared.

Why having Ebola gene sequences is helpful

Some Ebola diagnostic tests have been designed to detect areas that have mutated in the Ebola virus samples from this outbreak, raising the possibility these tests might be losing accuracy. One of the things Sabeti plans to do next is test whether that's actually the case.


Diagnosing Ebola can actually be more difficult than it might sound. The disease often looks like a lot of other feverish illnesses that can be common. And at a later stage, only some patients end up bleeding.

However, it's essential to know who has it as soon as possible, especially so that health-care workers can use appropriate procedures to prevent transmission to themselves and others. So accurate diagnostic tests are absolutely needed.

Researchers are also planning to study the mutations to see if any of them are affecting Ebola's recent behavior. The number of mutations found is completely normal, and it isn't necessarily the case that they'll have a big effect. But it's possible that something intriguing could turn up.

For example, this outbreak has had a higher transmission rate and lower death rate than others, and researchers are curious if any of these mutations are related to that. (Right now, social factors are thought to be the main causes of these two changes.)

"It sets the stage for the next few years of research that will reveal the differences between this virus and previous versions of Ebola virus," says Erica Ollmann Saphire, who researches Ebola and similar viruses at The Scripps Research Institute in La Jolla, California.

"My laboratory will be using this sequence information to understand the molecular effects of these mutations," she says. "We will also be looking at our pool of antibody therapeutics beyond ZMapp to ensure that candidate cocktails are optimally effective against these circulating strains."

Those working on other long-term projects involving vaccines should also find this information helpful.

The longer Ebola circulates, the more opportunities it has to change — possibly for the worse

Although Ebola's mutation rate itself isn't anything unusual, the longer it's circulating in people, the more chances it will have to randomly come up with a mutation that it will find beneficial — possibly to the detriment of human health.

"You never want to give a virus that kind of opportunity," Sabeti says. "We hope that this work opens up new doors for more people to work together to stop this virus now."

http://www.vox.com/2014/8/28/6071071/eb ... t=thursday


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