Jutta E Laiho1, Sami Oikarinen2, Maarit Oikarinen3, Pär G Larsson4, Virginia M Stone5, Didier Hober6, Steven Oberste7, Malin Flodström-Tullberg8, Jorma Isola9, Heikki Hyöty10. 1. Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: jutta.e.laiho@staff.uta.fi. 2. Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: sami.oikarinen@uta.fi. 3. Department of Virology, Medical School, University of Tampere, Tampere, Finland. Electronic address: maarit.oikarinen@uta.fi. 4. Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: par.larsson@ki.se. 5. Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: virginia.stone@ki.se. 6. Laboratoire de Virologie EA3610, Université Lille 2, CHRU, France. Electronic address: didier.hober@chru-lille.fr. 7. Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA. Electronic address: soberste@cdc.gov. 8. Department of Medicine HS, Karolinska Institutet, Stockholm, Sweden. Electronic address: malin.flodstrom-tullberg@ki.se. 9. Institute of Biosciences and Medical Technology, Tampere, Finland. Electronic address: jorma.isola@staff.uta.fi. 10. Department of Virology, Medical School, University of Tampere, Tampere, Finland; Fimlab Laboratories, Pirkanmaa Hospital District, Tampere, Finland. Electronic address: heikki.hyoty@uta.fi.
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. OBJECTIVES: 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. STUDY DESIGN: 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.
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. OBJECTIVES: 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. STUDY DESIGN: 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: Jutta E Laiho; Maarit Oikarinen; Sarah J Richardson; Gun Frisk; Julius Nyalwidhe; Tanya C Burch; Margaret A Morris; Sami Oikarinen; Alberto Pugliese; Francesco Dotta; Martha Campbell-Thompson; Jerry Nadler; Noel G Morgan; Heikki Hyöty Journal: J Clin Virol Date: 2016-02-02 Impact factor: 3.168
Authors: Niila V V Saarinen; Jutta E Laiho; Sarah J Richardson; Marie Zeissler; Virginia M Stone; Varpu Marjomäki; Tino Kantoluoto; Marc S Horwitz; Amirbabak Sioofy-Khojine; Anni Honkimaa; Minna M Hankaniemi; Malin Flodström-Tullberg; Heikki Hyöty; Vesa P Hytönen; Olli H Laitinen Journal: Sci Rep Date: 2018-01-08 Impact factor: 4.379
Authors: Sami Salmikangas; Jutta E Laiho; Kerttu Kalander; Mira Laajala; Anni Honkimaa; Iryna Shanina; Sami Oikarinen; Marc S Horwitz; Heikki Hyöty; Varpu Marjomäki Journal: Microorganisms Date: 2020-12-04