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Abstract

Background

The recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging.

Aim

To investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.

Methods

We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.

Results

Direct metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV.

Conclusions

This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

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/content/10.2807/1560-7917.ES.2018.23.50.1800228
2018-12-13
2019-06-17
http://instance.metastore.ingenta.com/content/10.2807/1560-7917.ES.2018.23.50.1800228
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References

  1. Papa A. Emerging arboviral human diseases in Southern Europe. J Med Virol. 2017;89(8):1315-22.  https://doi.org/10.1002/jmv.24803  PMID: 28252204 
  2. Gould E, Pettersson J, Higgs S, Charrel R, de Lamballerie X. Emerging arboviruses: Why today? One Health. 2017;4:1-13.  https://doi.org/10.1016/j.onehlt.2017.06.001  PMID: 28785601 
  3. Weaver SC, Reisen WK. Present and future arboviral threats. Antiviral Res. 2010;85(2):328-45.  https://doi.org/10.1016/j.antiviral.2009.10.008  PMID: 19857523 
  4. Thiberville S-D, Moyen N, Dupuis-Maguiraga L, Nougairede A, Gould EA, Roques P, et al. Chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy. Antiviral Res. 2013;99(3):345-70.  https://doi.org/10.1016/j.antiviral.2013.06.009  PMID: 23811281 
  5. World Health Organization (WHO). WHO publishes list of top emerging diseases likely to cause major epidemics.Geneva:WHO; 2017 Nov 10. [Accessed 26 Feb 2018]; Available from: http://www.who.int/medicines/ebola-treatment/WHO-list-of-top-emerging-diseases/en/
  6. Burt FJ, Chen W, Miner JJ, Lenschow DJ, Merits A, Schnettler E, et al. Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen. Lancet Infect Dis. 2017;17(4):e107-17.  https://doi.org/10.1016/S1473-3099(16)30385-1  PMID: 28159534 
  7. Mavalankar D, Shastri P, Bandyopadhyay T, Parmar J, Ramani KV. Increased mortality rate associated with chikungunya epidemic, Ahmedabad, India. Emerg Infect Dis. 2008;14(3):412-5.  https://doi.org/10.3201/eid1403.070720  PMID: 18325255 
  8. Vos T, Allen C, Arora M, Barber RM, Bhutta ZA, Brown A, et al. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388(10053):1545-602.  https://doi.org/10.1016/S0140-6736(16)31678-6  PMID: 27733282 
  9. Patterson J, Sammon M, Garg M. Dengue, Zika and Chikungunya: Emerging Arboviruses in the New World. West J Emerg Med. 2016;17(6):671-9.  https://doi.org/10.5811/westjem.2016.9.30904  PMID: 27833670 
  10. Burt FJ, Rolph MS, Rulli NE, Mahalingam S, Heise MT. Chikungunya: a re-emerging virus. Lancet. 2012;379(9816):662-71.  https://doi.org/10.1016/S0140-6736(11)60281-X  PMID: 22100854 
  11. Simmons CP, Farrar JJ, van Vinh Chau N, Wills B. Dengue. N Engl J Med. 2012;366(15):1423-32.  https://doi.org/10.1056/NEJMra1110265  PMID: 22494122 
  12. Furuya-Kanamori L, Liang S, Milinovich G, Soares Magalhaes RJ, Clements ACA, Hu W, et al. Co-distribution and co-infection of chikungunya and dengue viruses. BMC Infect Dis. 2016;16(1):84.  https://doi.org/10.1186/s12879-016-1417-2  PMID: 26936191 
  13. Perera-Lecoin M, Luplertlop N, Surasombatpattana P, Liégeois F, Hamel R, Thongrungkiat S, et al. Dengue and Chikungunya Coinfection – The Emergence of an Underestimated Threat. In: Rodriguez-Morales AJ, editor. Current Topics in Chikungunya. InTech; 2016.
  14. Omarjee R, Prat C, Flusin O, Boucau S, Tenebray B, Merle O, et al. Importance of case definition to monitor ongoing outbreak of chikungunya virus on a background of actively circulating dengue virus, St Martin, December 2013 to January 2014. Euro Surveill. 2014;19(13):20753.  https://doi.org/10.2807/1560-7917.ES2014.19.13.20753  PMID: 24721537 
  15. Brito CAA, Azevedo F, Cordeiro MT, Marques ETA Jr, Franca RFO. Central and peripheral nervous system involvement caused by Zika and chikungunya coinfection. PLoS Negl Trop Dis. 2017;11(7):e0005583.  https://doi.org/10.1371/journal.pntd.0005583  PMID: 28704365 
  16. Wilder-Smith A, Gubler DJ, Weaver SC, Monath TP, Heymann DL, Scott TW. Epidemic arboviral diseases: priorities for research and public health. Lancet Infect Dis. 2017;17(3):e101-6.  https://doi.org/10.1016/S1473-3099(16)30518-7  PMID: 28011234 
  17. Briese T, Paweska JT, McMullan LK, Hutchison SK, Street C, Palacios G, et al. Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa. PLoS Pathog. 2009;5(5):e1000455.  https://doi.org/10.1371/journal.ppat.1000455  PMID: 19478873 
  18. Towner JS, Sealy TK, Khristova ML, Albariño CG, Conlan S, Reeder SA, et al. Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 2008;4(11):e1000212.  https://doi.org/10.1371/journal.ppat.1000212  PMID: 19023410 
  19. Grard G, Fair JN, Lee D, Slikas E, Steffen I, Muyembe J-J, et al. A novel rhabdovirus associated with acute hemorrhagic fever in central Africa. PLoS Pathog. 2012;8(9):e1002924.  https://doi.org/10.1371/journal.ppat.1002924  PMID: 23028323 
  20. Metsky HC, Matranga CB, Wohl S, Schaffner SF, Freije CA, Winnicki SM, et al. Zika virus evolution and spread in the Americas. Nature. 2017;546(7658):411-5.  https://doi.org/10.1038/nature22402  PMID: 28538734 
  21. Grubaugh ND, Ladner JT, Kraemer MUG, Dudas G, Tan AL, Gangavarapu K, et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature. 2017;546(7658):401-5.  https://doi.org/10.1038/nature22400  PMID: 28538723 
  22. Faria NR, Quick J, Claro IM, Thézé J, de Jesus JG, Giovanetti M, et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature. 2017;546(7658):406-10.  https://doi.org/10.1038/nature22401  PMID: 28538727 
  23. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc. 2017;12(6):1261-76.  https://doi.org/10.1038/nprot.2017.066  PMID: 28538739 
  24. Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016;530(7589):228-32.  https://doi.org/10.1038/nature16996  PMID: 26840485 
  25. Walter MC, Zwirglmaier K, Vette P, Holowachuk SA, Stoecker K, Genzel GH, et al. MinION as part of a biomedical rapidly deployable laboratory. J Biotechnol. 2017;250:16-22.  https://doi.org/10.1016/j.jbiotec.2016.12.006  PMID: 27939320 
  26. Brown BL, Watson M, Minot SS, Rivera MC, Franklin RB. MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach. Gigascience. 2017;6(3):1-10.  https://doi.org/10.1093/gigascience/gix007  PMID: 28327976 
  27. Batovska J, Lynch SE, Rodoni BC, Sawbridge TI, Cogan NO. Metagenomic arbovirus detection using MinION nanopore sequencing. J Virol Methods. 2017;249:79-84.  https://doi.org/10.1016/j.jviromet.2017.08.019  PMID: 28855093 
  28. Greninger AL, Naccache SN, Federman S, Yu G, Mbala P, Bres V, et al. Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med. 2015;7(1):99.  https://doi.org/10.1186/s13073-015-0220-9  PMID: 26416663 
  29. Sardi SI, Somasekar S, Naccache SN, Bandeira AC, Tauro LB, Campos GS, et al. Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing. J Clin Microbiol. 2016;54(9):2348-53.  https://doi.org/10.1128/JCM.00877-16  PMID: 27413190 
  30. Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H, et al. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol. 2002;40(7):2323-30.  https://doi.org/10.1128/JCM.40.7.2323-2330.2002  PMID: 12089242 
  31. Edwards CJ, Welch SR, Chamberlain J, Hewson R, Tolley H, Cane PA, et al. Molecular diagnosis and analysis of Chikungunya virus. J Clin Virol. 2007;39(4):271-5.  https://doi.org/10.1016/j.jcv.2007.05.008  PMID: 17627877 
  32. Loman NJ, Quinlan AR. Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics. 2014;30(23):3399-401.  https://doi.org/10.1093/bioinformatics/btu555  PMID: 25143291 
  33. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754-60.  https://doi.org/10.1093/bioinformatics/btp324  PMID: 19451168 
  34. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. 1000 Genome Project Data Processing Subgroup. The Sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078-9.  https://doi.org/10.1093/bioinformatics/btp352  PMID: 19505943 
  35. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841-2.  https://doi.org/10.1093/bioinformatics/btq033  PMID: 20110278 
  36. Penedos AR, Myers R, Hadef B, Aladin F, Brown KE. Assessment of the Utility of Whole Genome Sequencing of Measles Virus in the Characterisation of Outbreaks. PLoS One. 2015;10(11):e0143081.  https://doi.org/10.1371/journal.pone.0143081  PMID: 26569100 
  37. Loman NJ, Quick J, Simpson JT. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods. 2015;12(8):733-5.  https://doi.org/10.1038/nmeth.3444  PMID: 26076426 
  38. Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15(3):R46.  https://doi.org/10.1186/gb-2014-15-3-r46  PMID: 24580807 
  39. Lewandowski K, Bell A, Miles R, Carne S, Wooldridge D, Manso C, et al. The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing. J Mol Diagn. 2017;19(2):303-12.  https://doi.org/10.1016/j.jmoldx.2016.10.005  PMID: 28041870 
  40. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-77.  https://doi.org/10.1089/cmb.2012.0021  PMID: 22506599 
  41. Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics. 2011;27(4):578-9.  https://doi.org/10.1093/bioinformatics/btq683  PMID: 21149342 
  42. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722-36.  https://doi.org/10.1101/gr.215087.116  PMID: 28298431 
  43. Andersen KG, Shapiro BJ, Matranga CB, Sealfon R, Lin AE, Moses LM, et al. Viral Hemorrhagic Fever Consortium. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus. Cell. 2015;162(4):738-50.  https://doi.org/10.1016/j.cell.2015.07.020  PMID: 26276630 
  44. Houldcroft CJ, Beale MA, Breuer J. Clinical and biological insights from viral genome sequencing. Nat Rev Microbiol. 2017;15(3):183-92.  https://doi.org/10.1038/nrmicro.2016.182  PMID: 28090077 
  45. Keita M, Duraffour S, Loman NJ, Rambaut A, Diallo B, Magassouba N, et al. Unusual Ebola Virus Chain of Transmission, Conakry, Guinea, 2014-2015. Emerg Infect Dis. 2016;22(12):2149-52.  https://doi.org/10.3201/eid2212.160847  PMID: 27869596 
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