title: Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells.
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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: Department of Virology, School of Medicine, 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, School of Medicine, University of Tampere, Tampere, Finland. Electronic address: maarit.oikarinen@uta.fi.
authors.forename: Maarit
authors.initials: M
authors.lastname: Oikarinen
authors.affiliation: University of Exeter Medical School, Exeter, Devon, UK. Electronic address: s.richardson@exeter.ac.uk.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Richardson
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address: Gun.Frisk@igp.uu.se.
authors.forename: Gun
authors.initials: G
authors.lastname: Frisk
authors.affiliation: 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.
authors.forename: Julius
authors.initials: J
authors.lastname: Nyalwidhe
authors.affiliation: 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.
authors.forename: Tanya C
authors.initials: TC
authors.lastname: Burch
authors.affiliation: Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, USA; Internal Medicine, Eastern Virginia Medical School, Norfolk, USA. Electronic address: MorrisMA@evms.edu.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Morris
authors.affiliation: Department of Virology, School of Medicine, University of Tampere, Tampere, Finland. Electronic address: sami.oikarinen@uta.fi.
authors.forename: Sami
authors.initials: S
authors.lastname: Oikarinen
authors.affiliation: 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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Pugliese
authors.affiliation: 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.
authors.forename: Francesco
authors.initials: F
authors.lastname: Dotta
authors.affiliation: University of Florida, Gainesville, Florida, USA. Electronic address: thompmc@pathology.ufl.edu.
authors.forename: Martha
authors.initials: M
authors.lastname: Campbell-Thompson
authors.affiliation: Internal Medicine, Eastern Virginia Medical School, Norfolk, USA. Electronic address: NadlerJL@EVMS.EDU.
authors.forename: Jerry
authors.initials: J
authors.lastname: Nadler
authors.affiliation: University of Exeter Medical School, Exeter, Devon, UK. Electronic address: n.g.morgan@exeter.ac.uk.
authors.forename: Noel G
authors.initials: NG
authors.lastname: Morgan
authors.affiliation: Department of Virology, School of Medicine, 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: D000072283:A549 Cells; D003384:Coxsackievirus Infections / Q000175:diagnosis / Q000821:virology; D029822:Enterovirus B, Human / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
keywords: Immunohistochemisrty; In situ hybridization; Proteomics; RT-PCR; Sensitivity