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Home Eurosurveillance Edition  2015: Volume 20/ Issue 40 Article 2
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Eurosurveillance, Volume 20, Issue 40, 08 October 2015
Rapid communication
Smits, Pas, Reusken, Haagmans, Pertile, Cancedda, Dierberg, Wurie, Kamara, Kargbo, Caddy, Arias, Thorne, Lu, Jah, Goodfellow, and Koopmans: Genotypic anomaly in Ebola virus strains circulating in Magazine Wharf area, Freetown, Sierra Leone, 2015

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Citation style for this article: Smits SL, Pas SD, Reusken CB, Haagmans BL, Pertile P, Cancedda C, Dierberg K, Wurie I, Kamara A, Kargbo D, Caddy SL, Arias A, Thorne L, Lu J, Jah U, Goodfellow I, Koopmans MP. Genotypic anomaly in Ebola virus strains circulating in Magazine Wharf area, Freetown, Sierra Leone, 2015. Euro Surveill. 2015;20(40):pii=30035. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2015.20.40.30035

Received:21 September 2015; Accepted:08 October 2015


In Sierra Leone, two new Ebola virus (EBOV) cases were reported from the densely populated Magazine Wharf area of Freetown in the Western Area Urban district after a period of two weeks in June 2015 with no cases in the district. The Magazine Wharf area was subsequently a focus of transmission for several weeks (http://apps.who.int/ebola/current-situation/ebola-situation-report-15-july-2015) up to 12 August 2015 (http://apps.who.int/ebola/current-situation/ebola-situation-report-12-august-2015), after which no new cases were reported from the area (http://apps.who.int/ebola/current-situation/ebola-situation-report-30-september-2015). In this study, the whole genomes of viruses from patient samples, originating from the Western Area Urban district and other districts of the country (i.e. Kenema, Kono, and Tonkolili) between January and July 2015 are sequenced. Genomes derived from samples collected from 30 June onwards in the Western Area Urban district have a particular anomaly consisting of a series of 13 T to C substitutions in a 150 bp intergenic region downstream of the viral protein 40 (VP40) open reading frame (ORF). This anomaly is also present in a viral strain, the Ebolavirus/H.sapiens-wt/SLE/2014/Makona-J0169 (J0169), which was detected in Freetown in November 2014. The finding suggests that viruses retrieved in June and July 2015 from the Western Area Urban district are direct descendants of a J0169-like virus. Near real time application of whole EBOV genome sequencing and the identification of lineage signatures can be used to monitor the ongoing outbreak and test whether newly infected patients are part of an identified transmission chain.

Ebola virus disease epidemic in West Africa

An epidemic of EBOV (a negative-sense RNA virus, family Filoviridae) disease has been ongoing in West Africa since December 2013 affecting mainly Guinea, Liberia and Sierra Leone [1]. As of 9 September 2015, the cumulative number of suspect, probable and confirmed cases stands at 28,183, including 11,306 deaths (http://apps.who.int/ebola/current-situation/ebola-situation-report-9-september-2015). EBOV cases continue to be detected and shifts in foci of transmission are observed. One of the pillars in the emergency response to the epidemic in the area has been the deployment of temporary (mobile) laboratories by the international community in collaboration with local authorities, among which the Dutch Mobile Laboratories (http://dutchebolalabs.nl/rapport-implementatie-dutch-mobile-labs-in-sierra-leone-en-liberia/). These laboratories provide rapid testing capacity for EBOV and malaria in support of clinical triage of (suspected) patients. In addition, the international community is currently working together in sequencing EBOV genomes in order to study EBOV evolution [2-7].

Sampling and whole genome sequencing

A total of 49 samples of EBOV positive patients who had been tested by Dutch Mobile Laboratories (Table) located in the Western Area Urban and Kono districts were included in the study. The samples were collected from patients residing in Kenema, Kono, Tonkolili and Western Area Urban districts between January and July 2015. Nucleic acids were extracted from the samples using EZ Advanced XL automated RNA extraction (Qiagen). Isolated nucleic acids were subjected to reverse transcription/polymerase chain reaction (PCR) amplification using the Ion AmpliSeq Ebola Panel Assay (Thermo Fisher Scientific) and the Ion Torrent sequencing platform at a local sequencing facility established at the Mateneh Ebola Treatment Centre in Makeni, Bombali district, Sierra Leone (European Nucleotide Archive Study: PRJEB10265). Reads were extracted from unfiltered BAM files using CLC Genomics Workbench 7.5.1 and trimmed based on quality with an ambiguous limit of 2 and quality limit of 0.05. Reads longer than 1,000 nucleotides (nt) or shorter than 15 nt were discarded. Trimmed reads were mapped to Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C05, complete genome (GenBank accession number: KJ660348), using CLC Genomics Workbench 7.5.1 map reads to reference beta module, with the following parameters: no masking, mismatch cost 2, insertion and deletion open cost 7, insertion and deletion extend cost 3, length fraction 0.95, similarity fraction 0.9. Consensus sequences were extracted and regions where depth of coverage was less than 2 were called as ‘N’. All generated genomes were manually inspected for accuracy, such as for the presence of intact ORFs and low coverage regions for adequate base calling, resulting in 48 near full-length EBOV genomes (Table).

Table

Characteristics of Ebola virus positive specimens, which were subjected to whole genome sequencing, Sierra Leone, January–July 2015 (n=49)

Specimen ID Specimen type Country Districta Date of specimen collection Accession number
12033 Blood Sierra Leone Western_Urban 19–02–2015 KT357813
12051 Blood Sierra Leone Western_Urban 21–02–2015 KT357814
12116 Blood Sierra Leone Western_Urban 26–02–2015 KT357815
12117 Blood Sierra Leone Western_Urban 26–02–2015 KT357816
12120 Blood Sierra Leone Western_Urban 27–02–2015 KT357817
12137 Blood Sierra Leone Western_Urban 28–02–2015 KT357819
12194 Blood Sierra Leone Western_Urban 04–03–2015 KT357818
12239 Swab Sierra Leone Western_Urban 07–03–2015 KT357820
12260 Blood Sierra Leone Western_Urban 09–03–2015 KT357821
12268 Blood Sierra Leone Western_Urban 10–03–2015 KT357822
12458 Blood Sierra Leone Western_Urban 28–03–2015 KT357823
12485b Swab Sierra Leone Western_Urban 31–03–2015 KT357824
24502b Blood Sierra Leone Kono 13–01–2015 KT357825
24504 Blood Sierra Leone Kono 13–01–2015 KT357826
24506 Blood Sierra Leone Kono 14–01–2015 KT357827
24511 Blood Sierra Leone Kono 14–01–2015 KT357828
24552 Blood Sierra Leone Kono 17–01–2015 KT357829
24553 Blood Sierra Leone Kono 17–01–2015 KT357830
24573 Swab Sierra Leone Kono 18–01–2015 KT357831
24581 Swab Sierra Leone Kono 19–01–2015 KT357832
24592* Swab Sierra Leone Kono 20–01–2015 KT357833
24601* Blood Sierra Leone Kono 20–01–2015 KT357834
24604 Blood Sierra Leone Kono 20–01–2015 KT357835
24605 Blood Sierra Leone Kono 20–01–2015 KT357836
24606 Blood Sierra Leone Kono 20–01–2015 KT357837
24608 Blood Sierra Leone Kono 20–01–2015 KT357838
24611 Swab Sierra Leone Kono 21–01–2015 KT357839
24620b Blood Sierra Leone Kono 21–01–2015 KT357840
24669 Blood Sierra Leone Kono 25–01–2015 KT357841
24677 Swab Sierra Leone Kono 25–01–2015 KT357842
24683** Blood Sierra Leone Kono 26–01–2015 KT357843
24706 Blood Sierra Leone Kono 28–01–2015 KT357844
24708**c Blood Sierra Leone Kono 28–01–2015 KT357845
24720b Blood Sierra Leone Kono 29–01–2015 KT357846
24758 Blood Sierra Leone Kono 30–01–2015 KT357847
24818 Swab Sierra Leone Kono 03–02–2015 KT357848
24825 Blood Sierra Leone Tonkolili 04–02–2015 KT357849
24853 Blood Sierra Leone Kono 06–02–2015 KT357850
24854 Blood Sierra Leone Kono 06–02–2015 KT357851
25083*** Blood Sierra Leone Kono 18–02–2015 KT357852
25103*** Swab Sierra Leone Kono 19–02–2015 KT357853
25123b Swab Sierra Leone Kenema 18–02–2015 KT357854
25180**** Blood Sierra Leone Kono 23–02–2015 KT357855
25344**** Blood Sierra Leone Kono 06–03–2015 KT357856
25411b Blood Sierra Leone Kono 10–03–2015 KT357857
13828 Blood Sierra Leone Western_Urban 22–06–2015 Not applicable
14077 Blood Sierra Leone Western_Urban 30–06–2015 KT357860
14163 Blood Sierra Leone Western_Urban 03–07–2015 KT357858
14366 Swab Sierra Leone Western_Urban 11–07–2015 KT357859

ID: identity; Western_Urban: Western Area Urban district.

In the column with specimen IDs, entries with the same number of asterisks indicate specimens derived from the same patient.

a District of patient residence.

b Less reliable consensus sequence due to low coverage regions.

c Not shown in Figure, unreliable consensus sequence due to low coverage regions.

Detection of evolutionary lineages and mutations

A median haplotype network was constructed in PopART version 1.7 (http://popart.otago.ac.nz) using 563 determined EBOV genomes from Sierra Leone, including those determined in this study ([3-5,7]; Figure A; Table). Accordingly, the 48 determined genome sequences appeared to belong to multiple distinct evolutionary lineages. Most viruses from the Western Area Urban district grouped together as did viruses from Kono.

Figure

A) Median-joining haplotype network constructed from an alignment of 563 Ebola virus sequences derived from clinical samples and B) alignment of a sequence region where four Ebola virus strains present a genotypical anomaly, Sierra Leone, 2015

/images/dynamic/articles/21270/15-00538-f1

GP: glycoprotein; L: RNA-dependent RNA polymerase L; NP: nucleoprotein; VP: virus protein; WUR: Western Area Urban district.

A: The 563 Ebola virus isolates’ sequences correspond to nucleotides 148–18,629 of Zaire ebolavirus isolate H.sapiens-wt/SLE/2015/Makona-Goderich1 (GenBank accession number: KT345616). The median haplotype network was constructed using PopART version 1.7 (http://popart.otago.ac.nz). Each vertex represents a sampled viral haplotype. The size of each vertex is relative to the number of sampled isolates. Coloured vertices indicate determined viral genomes from Western Area Urban (red), Kono (green), Tonkolili (blue), and Kenema (purple) districts. The larger font numbers depicted in red represent viral genomes derived from Freetown patients’ samples retrieved in June and July 2015 (two of them linked to Magazine Wharf area) with a genotypic anomaly consisting of a series of 13 T to C mutations in a 150 bp long sequence, which is located in an intergenic region downstream of the VP40 open reading frame. Hatch marks indicate single nucleotide mutations alongside each edge. The node labelled 24592 contains sequences 24592 and 24601; node 25103 contains 25083 and 25103; node 14163 contains 14163 and 14366; node 24552 contains 24552 and 24581; node 24854 contains 24854, 24853, 24605, 24669, 24677, 24604, and 24758; node 24825 contains 24825 and 24611; node 24818 contains 24818, 24553, and 24573.

B: Genotypic anomaly in four Ebola virus strains from Freetown, Western Area Urban district, consisting of a series of 13 T to C mutations in a 150 bp long sequence located in an intergenic region downstream of the VP40 open reading frame (genome positions: 5,510–5,633). Three of the strains, which are depicted as DML14077, DML14163, and DML14366 on the alignment were characterised in June and July 2015. These are respectively EBOV_DML14077_SLe_WesternUrban_20150630 (GenBank accession number: KT357860), EBOV_DML14163_SLe_WesternUrban_20150703 (GenBank accession number: KT357858), and EBOV_DML14366_SLe_WesternUrban_20150711 (GenBank accession number: KT357859). The fourth strain, which is shown as J0169 is Ebolavirus/H.sapiens-wt/SLE/2014/Makona-J0169 (GenBank accession number: KP759706) and was characterised in November 2014. The sequences of the four strains are compared with the reference Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C05, complete genome (GenBank accession number: KJ660348). The Ebola virus genome organisation is shown for reference.

Viruses EBOV_DML14077_SLe_WesternUrban_20150630, EBOV_DML14163_SLe_WesternUrban_20150703, and EBOV_DML14366_SLe_WesternUrban_20150711 (DML14077, DML14163, DML14366) isolated from patients from Freetown at the end of June and July (Table) were highly similar to each other and were clearly different from viruses isolated in Freetown between January and March 2015 (Figure A). Two of these viruses were linked to the Magazine Wharf area in Freetown, a hotspot for EBOV transmission in June and July 2015.

The DML14077, DML14163, and DML14366 viruses were most closely related to Ebolavirus/H.sapiens-wt/SLE/2014/Makona-J0169 (GenBank accession number: KP759706) isolated on 9 November 2014 in Freetown (Figure A). The J0169 virus had a striking genotypic anomaly containing a series of 13 T to C substitutions occurring in a region of 150 bp in length in an intergenic region downstream of the viral protein 40 (VP40) ORF. This anomaly was also present in DML14077, DML14163 and DML14366 (Figure B). An excessive accumulation of T to C mutations has been previously observed in a variety of virus genomes, including negative-sense RNA viruses and in EBOV in infected cells in vitro, and are a hallmark of host adenosine deaminases acting on RNA (ADARs) [8-10]. DML14077, DML14163, and DML14366 had 19, 17, and 18 additional single nt variants, respectively, compared with the J0169 virus, consistent with current estimates of evolutionary rate in the Ebola virus outbreak [5], suggesting that DML14077, DML14163 and DML14366 are direct descendants from a J0169-like virus (Figure A). The same stretch of T to C substitutions was found in a partial genome sequence from another patient at the end of June, who was also linked to the Magazine Wharf area (DML13828; Table; data not shown) and it serves as a lineage signature allowing identification and tracking of transmission chains.

Discussion and conclusions

At present only a handful of Ebola virus genomes sequenced from the current outbreak contain stretches of T to C mutations, and these can be found at different locations in the genome [5,7]. All shared nt variants, such as the conserved T to C mutations observed between J0169 [7] and the three viral genomes newly characterised in this study, are unlikely the source of recurrent induced mutations in individual patients. Rather, the T to C substitutions could have been fixed in viral lineages in the past and these lineages may be capable of efficient human-to-human transmission. This would explain how a J0169-like virus with a characteristic series of T to C mutations could have further spread in the Magazine Wharf area of Freetown.

It is unlikely that the T to C mutation stretch observed in the J0169-like viruses had a fitness advantage over other spreading EBOV lineages as in the ca 6–7 months of transmission between the first identification of this virus lineage (November 2014) and the present, this EBOV lineage did not become dominant over others. The J0169-like virus lineage seemingly lingered on at sufficiently low prevalence that it was not captured in genomic surveillance until now. This is not surprising given that the amount of whole genome sequences available is only a fraction of the 28,183 confirmed, probable and suspected cases (http://apps.who.int/ebola/current-situation/ebola-situation-report-9-september-2015) and whole genome sequencing started to build momentum relatively late in the outbreak. It shows however, that near real time application of whole EBOV genome sequencing and the identification of lineage signatures can be used to monitor the ongoing outbreak and test whether newly infected patients are part of an existing known transmission chain in the area.


Acknowledgements

We thank the hospital staff of PCMH, ETU, ODCH, and Koidu ETU for their efforts in the Ebola virus outbreak. We thank the Office of the President Sierra Leone, the Sierra Leone Ministry of Health and Sanitation Sierra Leone, and Partners in Health for continuous support and efforts in the Ebola virus outbreak response. We thank the drivers, pilots, non-governmental organisations, district medical and surveillance officers for their help with sample collection and logistics in Sierra Leone. We thank Bart Kooi, Adam Hume, Amber Hendriks, Anne van der Linden, Annelies Mesman, Annemieke Dinkla, Arie Kant, Bas van der Veer, Chantal Reusken, Cindy Warringa, Eline Verheij, Ellis Meuleman, Emily Nelson, Emre Kucukkose, Erik van Hannen, Evelien Kern, Felix Geeraedts, Florine Scholte, Gudrun Freidl, Guido van der Net, Hans Naus, Harry Vennema, Heidi De Gruyter, Hein Sprong, Heleen Klos, Henny Pieterse Bruins, Irma Verhoofstad, Jaco Verweij, Jacqueline Schelfaut, Janienne Klaasse, Janke Schinkel, Janko van Beek, Jelke Fros, Jennifer de Jong, Jeroen Cremer, Jeroen Roose, Jet Kant, Joffrey van Prehn, Jolanda Maaskant, Judy Yen, Jurre Siegers, Katja Wolthers, Lesla Bruijnesteijn, Lisa de Ruijter, Ludo Oostendorp, Marion Nederpel, Marion Sunter, Marloes Dullaart, Martine Slop, Matthew McCall, Nancy Beerens, Naomi Berkeveld, Nathalie van Burgel, Nicole van Manen, Nienke van Teijlingen, Nina Hertoghs, Petra van den Doel, Pieter Smit, Richard de Boer, Robbert Bentvelsen, Robin van Houdt, Ruud Jansen, Saskia Bierman, Seta Jahfari, Stefan Boers, Stephanie Blanc, Suzan Pas, and Viviana Cobos for operating the Dutch Mobile Laboratories. We thank all other people involved in getting the Dutch Mobile Laboratories up and running. We thank Terry Jones and Matt Cotton for support in transferring sequence data to the Netherlands. We thank Andrew Rambaut for helpful discussions in preparing this manuscript. This work was supported by ZonMW TOP project 91213058, the European Union’s Horizon 2020 research and innovation programme under grant agreement No 643476 (COMPARE), and the Wellcome trust under grant 097997/Z/11/A.

Conflict of interest

None declared.

Authors’ contributions

SLS, SDP, CBR, BLH and MPK were involved in data analysis, training of people operating Dutch Mobile Laboratories in Sierra Leone, and writing the manuscript. PP, CC, KD, IW, AK, and DK were involved in getting Dutch Mobile Laboratories operational and providing continuous support in gathering clinical samples. SLC, AA, LT, JL, UJ, and IG were involved in next generation sequencing in Sierra Leone and writing the manuscript.


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