title: Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology.
abstract: BACKGROUND
  Enteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype.
  
  
  OBJECTIVE
  The aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples.
  
  
  METHODS
  Four enterovirus RNA-targeted oligonucleotide RNA probes - two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) - were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata.
  
  
  RESULTS
  The two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB -infected mice.
  
  
  CONCLUSIONS
  These novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.
authors.affiliation: Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: jutta.e.laiho@staff.uta.fi.
authors.forename: Jutta E
authors.initials: JE
authors.lastname: Laiho
authors.affiliation: Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: sami.oikarinen@uta.fi.
authors.forename: Sami
authors.initials: S
authors.lastname: Oikarinen
authors.affiliation: Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: maarit.oikarinen@uta.fi.
authors.forename: Maarit
authors.initials: M
authors.lastname: Oikarinen
authors.affiliation: Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: par.larsson@ki.se.
authors.forename: Pär G
authors.initials: PG
authors.lastname: Larsson
authors.affiliation: Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: virginia.stone@ki.se.
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Stone
authors.affiliation: Laboratoire de Virologie EA3610, Université Lille 2, CHRU, France. Electronic address: didier.hober@chru-lille.fr.
authors.forename: Didier
authors.initials: D
authors.lastname: Hober
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address: soberste@cdc.gov.
authors.forename: Steven
authors.initials: S
authors.lastname: Oberste
authors.affiliation: Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: malin.flodstrom-tullberg@ki.se.
authors.forename: Malin
authors.initials: M
authors.lastname: Flodström-Tullberg
authors.affiliation: Institute of Biosciences and Medical Technology, Tampere, Finland. Electronic address: jorma.isola@staff.uta.fi.
authors.forename: Jorma
authors.initials: J
authors.lastname: Isola
authors.affiliation: Department of Virology, Medical School, University of Tampere, Tampere, Finland; Fimlab Laboratories, Pirkanmaa Hospital District, Tampere, Finland. Electronic address: heikki.hyoty@uta.fi.
authors.forename: Heikki
authors.initials: H
authors.lastname: Hyöty
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D019295:Computational Biology / Q000379:methods; D004770:Enterovirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D025202:Molecular Diagnostic Techniques / Q000379:methods; D010179:Pancreas / Q000821:virology; D015347:RNA Probes / Q000032:analysis*; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D014709:Vero Cells
chemical_list: D015347:RNA Probes; D012367:RNA, Viral
keywords: Enterovirus; In situ hybridization; Probe design; Type 1 diabetes