title: Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA.
abstract: BACKGROUND
  The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures.
  
  
  METHODS
  VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers.
  
  
  RESULTS
  Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community.
  
  
  CONCLUSIONS
  Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.
authors.forename: Shahzad
authors.initials: S
authors.lastname: Shaukat
authors.forename: Mehar
authors.initials: M
authors.lastname: Angez
authors.forename: Muhammad Masroor
authors.initials: MM
authors.lastname: Alam
authors.forename: Maarten F
authors.initials: MF
authors.lastname: Jebbink
authors.forename: Martin
authors.initials: M
authors.lastname: Deijs
authors.forename: Marta
authors.initials: M
authors.lastname: Canuti
authors.forename: Salmaan
authors.initials: S
authors.lastname: Sharif
authors.forename: Michel
authors.initials: M
authors.lastname: de Vries
authors.forename: Adnan
authors.initials: A
authors.lastname: Khurshid
authors.forename: Tariq
authors.initials: T
authors.lastname: Mahmood
authors.forename: Lia
authors.initials: L
authors.lastname: van der Hoek
authors.affiliation: Department of Virology, National Institute of Health, Chak Shahzad, Park Road, Islamabad 45500, Pakistan. sohailz@live.com.
authors.forename: Syed Sohail Zahoor
authors.initials: SS
authors.lastname: Zaidi
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods; D036022:Capsid Proteins / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D021821:Communicable Diseases, Emerging / Q000175:diagnosis / Q000821:virology*; D005243:Feces / Q000821:virology*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D008969:Molecular Sequence Data; D010264:Paraplegia / Q000821:virology*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D014777:Virus Diseases / Q000175:diagnosis / Q000821:virology*; D014780:Viruses / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D036022:Capsid Proteins
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