Jutta E Laiho1, Maarit Oikarinen2, Sarah J Richardson3, Gun Frisk4, Julius Nyalwidhe5, Tanya C Burch6, Margaret A Morris7, Sami Oikarinen8, Alberto Pugliese9, Francesco Dotta10, Martha Campbell-Thompson11, Jerry Nadler12, Noel G Morgan13, Heikki Hyöty14. 1. Department of Virology, School of Medicine, University of Tampere, Tampere, Finland. Electronic address: jutta.e.laiho@staff.uta.fi. 2. Department of Virology, School of Medicine, University of Tampere, Tampere, Finland. Electronic address: maarit.oikarinen@uta.fi. 3. University of Exeter Medical School, Exeter, Devon, UK. Electronic address: s.richardson@exeter.ac.uk. 4. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address: Gun.Frisk@igp.uu.se. 5. Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, USA; Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, USA. Electronic address: NyalwiJO@evms.edu. 6. Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, USA; Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, USA. Electronic address: BurchTC@EVMS.EDU. 7. Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, USA; Internal Medicine, Eastern Virginia Medical School, Norfolk, USA. Electronic address: MorrisMA@evms.edu. 8. Department of Virology, School of Medicine, University of Tampere, Tampere, Finland. Electronic address: sami.oikarinen@uta.fi. 9. Diabetes Research Institute and Departments of Medicine, Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, USA. Electronic address: APuglies@med.miami.edu. 10. Diabetes Unit, Dept. of Medicine Surgery and Neurosciences, University of Siena; Fondazione Umberto Di Mario ONLUS-Toscana Life Sciences, Siena, Italy,. Electronic address: Francesco.dotta@alice.it. 11. University of Florida, Gainesville, Florida, USA. Electronic address: thompmc@pathology.ufl.edu. 12. Internal Medicine, Eastern Virginia Medical School, Norfolk, USA. Electronic address: NadlerJL@EVMS.EDU. 13. University of Exeter Medical School, Exeter, Devon, UK. Electronic address: n.g.morgan@exeter.ac.uk. 14. Department of Virology, School of Medicine, University of Tampere, Tampere, Finland; Fimlab Laboratories, Pirkanmaa Hospital District, Tampere, Finland. Electronic address: heikki.hyoty@uta.fi.
Abstract
BACKGROUND: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. OBJECTIVES: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. STUDY DESIGN: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10(-1) to 10(-8). Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. RESULTS: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10(-8)). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10(-7). The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). CONCLUSIONS: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.
BACKGROUND: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. OBJECTIVES: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected humanA549 cells in vitro. STUDY DESIGN:A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10(-1) to 10(-8). Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. RESULTS: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10(-8)). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10(-7). The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). CONCLUSIONS: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.
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Authors: Jessica L Dunne; Sarah J Richardson; Mark A Atkinson; Maria E Craig; Knut Dahl-Jørgensen; Malin Flodström-Tullberg; Heikki Hyöty; Richard E Lloyd; Noel G Morgan; Alberto Pugliese Journal: Diabetologia Date: 2019-04-23 Impact factor: 10.122
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