Research article Open Access
Like 0


An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.


Article metrics loading...

Loading full text...

Full text loading...



  1. Centers for Disease Control and Prevention (CDC). Evaluating surveillance indicators supporting the Global Polio Eradication Initiative, 2011-2012. MMWR Morb Mortal Wkly Rep. 2013;62(14):270-4. PMID: 23575241 
  2. Hovi T, Shulman LM, van der Avoort H, Deshpande J, Roivainen M, DE Gourville EM. Role of environmental poliovirus surveillance in global polio eradication and beyond. Epidemiol Infect. 2012;140(1):1-13.  https://doi.org/10.1017/S095026881000316X  PMID: 21849095 
  3. Asghar H, Diop OM, Weldegebriel G, Malik F, Shetty S, El Bassioni L, et al. Environmental surveillance for polioviruses in the Global Polio Eradication Initiative. J Infect Dis. 2014;210(Suppl 1):S294-303.  https://doi.org/10.1093/infdis/jiu384  PMID: 25316848 
  4. Manor Y, Handsher R, Halmut T, Neuman M, Bobrov A, Rudich H, et al. Detection of poliovirus circulation by environmental surveillance in the absence of clinical cases in Israel and the Palestinian authority. J Clin Microbiol. 1999;37(6):1670-5. PMID: 10325305 
  5. Shulman LM, Gavrilin E, Jorba J, Martin J, Burns CC, Manor Y, et al. Molecular epidemiology of silent introduction and sustained transmission of wild poliovirus type 1, Israel, 2013. Euro Surveill. 2014;19(7):20709.  https://doi.org/10.2807/1560-7917.ES2014.19.7.20709  PMID: 24576471 
  6. El Bassioni L, Barakat I, Nasr E, de Gourville EM, Hovi T, Blomqvist S, et al. Prolonged detection of indigenous wild polioviruses in sewage from communities in Egypt. Am J Epidemiol. 2003;158(8):807-15.  https://doi.org/10.1093/aje/kwg202  PMID: 14561671 
  7. Shulman LM, Manor Y, Sofer D, Handsher R, Swartz T, Delpeyroux F, et al. Neurovirulent vaccine-derived polioviruses in sewage from highly immune populations. PLoS One. 2006;1(1):e69.  https://doi.org/10.1371/journal.pone.0000069  PMID: 17183700 
  8. Al-Hello H, Jorba J, Blomqvist S, Raud R, Kew O, Roivainen M. Highly divergent type 2 and 3 vaccine-derived polioviruses isolated from sewage in Tallinn, Estonia. J Virol. 2013;87(23):13076-80.  https://doi.org/10.1128/JVI.01174-13  PMID: 24049178 
  9. Hovi T, Paananen A, Blomqvist S, Savolainen-Kopra C, Al-Hello H, Smura T, et al. Characteristics of an environmentally monitored prolonged type 2 vaccine derived poliovirus shedding episode that stopped without intervention. PLoS One. 2013;8(7):e66849.  https://doi.org/10.1371/journal.pone.0066849  PMID: 23935826 
  10. Albertson P. Two-phase separation of viruses. In Maramorosch K, Koprowski H, eds. Methods of virology. 1967:303-21.
  11. Más Lago P, Gary HE Jr, Pérez LS, Cáceres V, Olivera JB, Puentes RP, et al. Poliovirus detection in wastewater and stools following an immunization campaign in Havana, Cuba. Int J Epidemiol. 2003;32(5):772-7.  https://doi.org/10.1093/ije/dyg185  PMID: 14559748 
  12. World Health Organization (WHO). Guidelines for environmental surveillance of poliovirus circulation. Geneva, Switzerland: WHO; 2003.
  13. Tambini G, Andrus JK, Marques E, Boshell J, Pallansch M, de Quadros CA, et al. Direct detection of wild poliovirus circulation by stool surveys of healthy children and analysis of community wastewater. J Infect Dis. 1993;168(6):1510-4.  https://doi.org/10.1093/infdis/168.6.1510  PMID: 8245537 
  14. Lewis GD, Metcalf TG. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol. 1988;54(8):1983-8. PMID: 2845860 
  15. Shields PA, Farrah SR. Concentration of viruses in beef extract by flocculation with ammonium sulfate. Appl Environ Microbiol. 1986;51(1):211-3. PMID: 3006587 
  16. Guttman-Bass N, Catalano-Sherman J. Humic acid interference with virus recovery by electropositive microporous filters. Appl Environ Microbiol. 1986;52(3):556-61. PMID: 3021058 
  17. Liu P, Hill VR, Hahn D, Johnson TB, Pan Y, Jothikumar N, et al. Hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water. J Microbiol Methods. 2012;88(1):155-61.  https://doi.org/10.1016/j.mimet.2011.11.007  PMID: 22108496 
  18. Smith CM, Hill VR. Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl Environ Microbiol. 2009;75(16):5284-9.  https://doi.org/10.1128/AEM.00456-09  PMID: 19561183 
  19. Thurber RV, Haynes M, Breitbart M, Wegley L, Rohwer F. Laboratory procedures to generate viral metagenomes. Nat Protoc. 2009;4(4):470-83.  https://doi.org/10.1038/nprot.2009.10  PMID: 19300441 
  20. Pipkin PA, Wood DJ, Racaniello VR, Minor PD. Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro. J Virol Methods. 1993;41(3):333-40.  https://doi.org/10.1016/0166-0934(93)90022-J  PMID: 8386181 
  21. Kilpatrick DR, Yang CF, Ching K, Vincent A, Iber J, Campagnoli R, et al. Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues. J Clin Microbiol. 2009;47(6):1939-41.  https://doi.org/10.1128/JCM.00702-09  PMID: 19386844 
  22. Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM, et al. Functional metagenomic profiling of nine biomes. Nature. 2008;452(7187):629-32.  https://doi.org/10.1038/nature06810  PMID: 18337718 
  23. Cantalupo PG, Calgua B, Zhao G, Hundesa A, Wier AD, Katz JP, et al. Raw sewage harbors diverse viral populations. MBio. 2011;2(5):e00180-11.  https://doi.org/10.1128/mBio.00180-11  PMID: 21972239 
  24. Ng TF, Marine R, Wang C, Simmonds P, Kapusinszky B, Bodhidatta L, et al. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. J Virol. 2012;86(22):12161-75.  https://doi.org/10.1128/JVI.00869-12  PMID: 22933275 
  25. Prachayangprecha S, Schapendonk CM, Koopmans MP, Osterhaus AD, Schürch AC, Pas SD, et al. Exploring the potential of next-generation sequencing in detection of respiratory viruses. J Clin Microbiol. 2014;52(10):3722-30.  https://doi.org/10.1128/JCM.01641-14  PMID: 25100822 
  26. Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, et al. RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol. 2006;4(1):e3.  https://doi.org/10.1371/journal.pbio.0040003  PMID: 16336043 
  27. Mueller C, Flotte TR. Clinical gene therapy using recombinant adeno-associated virus vectors. Gene Ther. 2008;15(11):858-63.  https://doi.org/10.1038/gt.2008.68  PMID: 18418415 
  28. Plotkin SA, Vidor E. Poliovirus vaccine -- inactivated. In: Plotkin SA, Orenstein WA, Offit PA, editors. Vaccines. 5 ed. Philadelphia: W.B. Saunders; 2008. p. 605-29.

Data & Media loading...

Submit comment
Comment moderation successfully completed
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error