Volume 95, Issue 3
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



Numerous pathogens cause respiratory infections with similar symptoms. Routine diagnostics detect only a limited number of pathogens, leaving a gap in respiratory illness etiology surveillance. This study evaluated next-generation sequencing for unbiased pathogen identification. Respiratory samples collected in Thailand, Philippines, Bhutan, and Nepal, that were negative by several molecular and immunofluorescence assays, underwent viral cultivation. Samples which demonstrated cytopathic effect in culture ( = 121) were extracted and tested by Luminex xTAG respiratory viral panel (RVP) assay and deep sequencing by Roche 454 FLX Titanium system. Using RVP assay, 52 (43%) samples were positive for enterovirus or rhinovirus and another three were positive for respiratory syncytial virus B, parainfluenza 4, and adenovirus. Deep sequencing confirmed the Luminex assay results and identified additional viral pathogens. Human enteroviruses, including type 71 and 12 types of (EV-B) were identified from a hospital in Bangkok. Phylogenetic and recombination analysis showed high correlation of VP1 gene-based phylogeny with genome-wide phylogeny and the frequent genetic exchange among EV-B viruses. The high number and diversity of enteroviruses in the hospital in Bangkok suggests prevalent existence. The metagenomic approach used in our study enabled comprehensive diagnoses of respiratory viruses.

[open-access] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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  1. Burke RL, Vest KG, Eick AA, Sanchez JL, Johns MC, Pavlin JA, Jarman RG, Mothershead JL, Quintana M, Palys T, Cooper MJ, Guan J, Schnabel D, Waitumbi J, Wilma A, Daniels C, Brown ML, Tobias S, Kasper MR, Williams M, Tjaden JA, Oyofo B, Styles T, Blair PJ, Hawksworth A, Montgomery JM, Razuri H, Laguna-Torres A, Schoepp RJ, Norwood DA, Macintosh VH, Gibbons T, Gray GC, Blazes DL, Russell KL, AFHSC-GEIS Influenza Surveillance Writing Group Rubenstein J, Hathaway K, Gibbons R, Yoon IK, Saunders D, Gaywee J, Stoner M, Timmermans A, Shrestha SK, Velasco JM, Alera MT, Tannitisupawong D, Myint KS, Pichyangkul S, Woods B, Jerke KH, Koenig MG, Byarugaba DK, Mangen FW, Assefa B, Williams M, Brice G, Mansour M, Pimentel G, Sebeny P, Talaat M, Saeed T, Espinosa B, Faix D, Maves R, Kochel T, Smith J, Guerrero A, Maupin G, Sjoberg P, Duffy M, Garner J, Canas L, Macias E, Kuschner RA, Shanks D, Lewis S, Nowak G, Ndip LM, Wolfe N, Saylors K, ; , , 2011. Department of Defense influenza and other respiratory disease surveillance during the 2009 pandemic. BMC Public Health 11 (Suppl 2): S6. [Google Scholar]
  2. Nguyen-Van-Tam JS, Sellwood C, , 2013. Intervention strategies for emerging respiratory virus infections: policy and public health considerations. Curr Opin Virol 3: 192198.[Crossref] [Google Scholar]
  3. Lipkin WI, , 2013. The changing face of pathogen discovery and surveillance. Nat Rev Microbiol 11: 133141.[Crossref] [Google Scholar]
  4. Jartti T, Jartti L, Ruuskanen O, Soderlund-Venermo M, , 2012. New respiratory viral infections. Curr Opin Pulm Med 18: 271278.[Crossref] [Google Scholar]
  5. Lipkin WI, , 2010. Microbe hunting. Microbiology and molecular biology reviews. Microbiol Mol Biol Rev 74: 363377.[Crossref] [Google Scholar]
  6. Fischer N, Indenbirken D, Meyer T, Lutgehetmann M, Lellek H, Spohn M, Aepfelbacher M, Alawi M, Grundhoff A, , 2015. Evaluation of unbiased next-generation sequencing of RNA (RNA-seq) as a diagnostic method in influenza virus-positive respiratory samples. J Clin Microbiol 53: 22382250.[Crossref] [Google Scholar]
  7. Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J, Tammadoni S, Nosrat B, Conrad D, Rohwer F, , 2009. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS One 4: e7370.[Crossref] [Google Scholar]
  8. Radford AD, Chapman D, Dixon L, Chantrey J, Darby AC, Hall N, , 2012. Application of next-generation sequencing technologies in virology. J Gen Virol 93: 18531868.[Crossref] [Google Scholar]
  9. Barzon L, Lavezzo E, Militello V, Toppo S, Palu G, , 2011. Applications of next-generation sequencing technologies to diagnostic virology. Int J Mol Sci 12: 78617884.[Crossref] [Google Scholar]
  10. Thorburn F, Bennett S, Modha S, Murdoch D, Gunson R, Murcia PR, , 2015. The use of next generation sequencing in the diagnosis and typing of respiratory infections. J Clin Virol 69: 96100.[Crossref] [Google Scholar]
  11. Muehlenbachs A, Bhatnagar J, Zaki SR, , 2014. Tissue tropism, pathology and pathogenesis of enterovirus infection. J Pathol 235: 217228.[Crossref] [Google Scholar]
  12. Stephenson J, , 2014. CDC tracking enterovirus D-68 outbreak causing severe respiratory illness in children in the Midwest. JAMA 312: 1290.[Crossref] [Google Scholar]
  13. Jubelt B, Lipton HL, , 2014. Enterovirus/picornavirus infections. Handb Clin Neurol 123: 379416.[Crossref] [Google Scholar]
  14. Hang J, Forshey BM, Kochel TJ, Li T, Solorzano VF, Halsey ES, Kuschner RA, , 2012. Random amplification and pyrosequencing for identification of novel viral genome sequences. J Biomol Tech 23: 410.[Crossref] [Google Scholar]
  15. Kroneman A, Vennema H, Deforche K, van der Avoort H, Penaranda S, Oberste MS, Vinje J, Koopmans M, , 2011. An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol 51: 121125.[Crossref] [Google Scholar]
  16. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, , 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 27252729.[Crossref] [Google Scholar]
  17. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P, , 2010. RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26: 24622463.[Crossref] [Google Scholar]
  18. Tavakoli NP, Tobin EH, Wong SJ, Dupuis AP, 2nd Glasheen B, Kramer LD, Bernard KA, , 2007. Identification of dengue virus in respiratory specimens from a patient who had recently traveled from a region where dengue virus infection is endemic. J Clin Microbiol 45: 15231527.[Crossref] [Google Scholar]
  19. Linsuwanon P, Puenpa J, Huang SW, Wang YF, Mauleekoonphairoj J, Wang JR, Poovorawan Y, , 2014. Epidemiology and seroepidemiology of human enterovirus 71 among Thai populations. J Biomed Sci 21: 16.[Crossref] [Google Scholar]
  20. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH, , 2010. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10: 778790.[Crossref] [Google Scholar]
  21. Zhang Y, Tan X, Cui A, Mao N, Xu S, Zhu Z, Zhou J, Shi J, Zhao Y, Wang X, Huang X, Zhu S, Zhang Y, Tang W, Ling H, Xu W, , 2013. Complete genome analysis of the C4 subgenotype strains of enterovirus 71: predominant recombination C4 viruses persistently circulating in China for 14 years. PLoS One 8: e56341.[Crossref] [Google Scholar]
  22. Wang Y, Zhu Q, Zeng M, Altmeyer R, Zou G, , 2014. Complete genome sequence of a human enterovirus 71 strain isolated from a fatal case in Shanghai, China, in 2012. Genome Announc 2: e00457-14.[Crossref] [Google Scholar]
  23. Oberste MS, Maher K, Pallansch MA, , 2004. Evidence for frequent recombination within species human enterovirus B based on complete genomic sequences of all thirty-seven (sero)types. J Virol 78: 855867.[Crossref] [Google Scholar]
  24. McVean G, Auton A, , 2007. LDhat 2.1: A Package for the Population Genetic Analysis of Recombination. Oxford, United Kingdom: Department of Statistics. Accessed at: http://www.stats.ox.ac.uk/∼mcvean/LDhat/manual.pdf. [Google Scholar]
  25. Yozwiak NL, Skewes-Cox P, Stenglein MD, Balmaseda A, Harris E, DeRisi JL, , 2012. Virus identification in unknown tropical febrile illness cases using deep sequencing. PLoS Negl Trop Dis 6: e1485.[Crossref] [Google Scholar]
  26. Wilson MR, Naccache SN, Samayoa E, Biagtan M, Bashir H, Yu G, Salamat SM, Somasekar S, Federman S, Miller S, Sokolic R, Garabedian E, Candotti F, Buckley RH, Reed KD, Meyer TL, Seroogy CM, Galloway R, Henderson SL, Gern JE, DeRisi JL, Chiu CY, , 2014. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med 370: 24082417.[Crossref] [Google Scholar]
  27. Barzon L, Lavezzo E, Costanzi G, Franchin E, Toppo S, Palu G, , 2013. Next-generation sequencing technologies in diagnostic virology. J Clin Virol 58: 346350.[Crossref] [Google Scholar]
  28. Huaman JL, Carrion G, Ampuero JS, Gomez J, Ocana V, Paz I, Gomez E, Chavez E, Sarmiento F, Pozo E, Laguna-Torres VA, Halsey ES, , 2014. Non-rhinovirus enteroviruses associated with respiratory infections in Peru (2005–2010). Virol J 11: 169.[Crossref] [Google Scholar]
  29. Zwaans WA, Mallia P, van Winden ME, Rohde GG, , 2014. The relevance of respiratory viral infections in the exacerbations of chronic obstructive pulmonary disease: a systematic review. J Clin Virol 61: 181188.[Crossref] [Google Scholar]
  30. Chan YF, Sam IC, AbuBakar S, , 2010. Phylogenetic designation of enterovirus 71 genotypes and subgenotypes using complete genome sequences. Infect Genet Evol 10: 404412.[Crossref] [Google Scholar]
  31. Garcia J, Espejo V, Nelson M, Sovero M, Villaran MV, Gomez J, Barrantes M, Sanchez F, Comach G, Arango AE, Aguayo N, de Rivera IL, Chicaiza W, Jimenez M, Aleman W, Rodriguez F, Gonzales MS, Kochel TJ, Halsey ES, , 2013. Human rhinoviruses and enteroviruses in influenza-like illness in Latin America. Virol J 10: 305.[Crossref] [Google Scholar]
  32. Richter J, Tryfonos C, Panagiotou C, Nikolaou E, Koliou M, Christodoulou C, , 2013. Newly emerging C group enteroviruses may elude diagnosis due to a divergent 5′-UTR. Int J Infect Dis 17: e1245e1248.[Crossref] [Google Scholar]
  33. Nix WA, Oberste MS, Pallansch MA, , 2006. Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus (sero)types from original clinical specimens. J Clin Microbiol 44: 26982704.[Crossref] [Google Scholar]
  34. Bessaud M, Razafindratsimandresy R, Nougairede A, Joffret ML, Deshpande JM, Dubot-Peres A, Heraud JM, de Lamballerie X, Delpeyroux F, Bailly JL, , 2014. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS One 9: e90624.[Crossref] [Google Scholar]

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  • Received : 28 Jan 2016
  • Accepted : 12 May 2016
  • Published online : 07 Sep 2016

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