Introduction. Virus detection and/or identification is traditionally performed using cell culture, electron microscopy and antigen or nucleic acid detection. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), commonly used in clinical microbiology, was developed and tested as an innovative tool to be applied to virus identification by using two different approaches. Materials and Methods. In the first approach, human polioviruses were selected as a model to evaluate the ability of MALDI-TOF MS to identify specific viral protein to be used as biomarkers of purified virus particles, followed by the serotypes identification. To this aim the Sabin reference strains (I, II, III) were firstly analysed and, subsequently, the results were then confirmed by a blind application of the assay to clinically isolated strains. In the second approach, a protein profiles library was newly create to discriminate between uninfected and respiratory virus infected cell cultures after a viral proteins enrichment method. The library was built using different reference strains after an extensive modification of the MALDI- TOF MS pre-processing, MSP creation, subtyping MSP creation and identification default parameters setting. Results. The very efficient technique adopted to obtain highly purified poliovirus allowed us to discriminate viral protein peaks from uninfected cells peaks and to detect specific poliovirus protein biomarkers. Moreover, MALDI-TOF MS analysis applied to the three Sabin poliovirus serotypes revealed characteristic peak profiles for each of them showing three independent clusters for the three serotypes. After a proper statistical investigation, the VP4 was used as a potential biomarker to identify poliovirus strains at the serotype level. On the bases of VP4 all clinical isolates were identified at the serotype level. In the second approach, the spectra generated from virus infected cell cultures revealed the presence of some different peaks not overlap- ping those of uninfected cell cultures for all the reference virus infected cell cultures. The parameters for the creation of the Main Spectrum Profile (MSP) for each of the reference virus infected cell cultures were set on the basis of these peaks. The obtained MSP spectra were used to create a new respiratory viruses library in our Bruker Daltonics database in order to blind identify viruses isolated from biological samples aster a cell culture step. The spectra obtained by 58 additional cultured strains correctly match with the new database demonstrating its reliability. Discussion and Conclusions. In conclusion, this study could be considered a starting point for further evolutions of the developed system, since the differences observed comparing the spectra obtained from virus infected cell culture suggest the possibility to apply these approaches to the identification of other viruses including other picornavirus such as enterovirus and coxsackievirus and viruses responsible for respiratory infections, as well as to viral agents causing infections of other body sites.

An innovative application of MALDI-TOF MS in clinical virology / Calderaro, Adriana; Buttrini, Mirko; Montecchini, Sara; Motta, Federica; Rodighiero, Isabella; Arcangeletti, Maria Cristina; Chezzi, Carlo; DE CONTO, Flora. - (2018), p. 153. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo nel 26-29 settembre2018.

An innovative application of MALDI-TOF MS in clinical virology

Adriana Calderaro;Mirko Buttrini;Sara Montecchini;Federica Motta;Isabella Rodighiero;Maria Cristina Arcangeletti;Carlo Chezzi;Flora De Conto
2018

Abstract

Introduction. Virus detection and/or identification is traditionally performed using cell culture, electron microscopy and antigen or nucleic acid detection. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), commonly used in clinical microbiology, was developed and tested as an innovative tool to be applied to virus identification by using two different approaches. Materials and Methods. In the first approach, human polioviruses were selected as a model to evaluate the ability of MALDI-TOF MS to identify specific viral protein to be used as biomarkers of purified virus particles, followed by the serotypes identification. To this aim the Sabin reference strains (I, II, III) were firstly analysed and, subsequently, the results were then confirmed by a blind application of the assay to clinically isolated strains. In the second approach, a protein profiles library was newly create to discriminate between uninfected and respiratory virus infected cell cultures after a viral proteins enrichment method. The library was built using different reference strains after an extensive modification of the MALDI- TOF MS pre-processing, MSP creation, subtyping MSP creation and identification default parameters setting. Results. The very efficient technique adopted to obtain highly purified poliovirus allowed us to discriminate viral protein peaks from uninfected cells peaks and to detect specific poliovirus protein biomarkers. Moreover, MALDI-TOF MS analysis applied to the three Sabin poliovirus serotypes revealed characteristic peak profiles for each of them showing three independent clusters for the three serotypes. After a proper statistical investigation, the VP4 was used as a potential biomarker to identify poliovirus strains at the serotype level. On the bases of VP4 all clinical isolates were identified at the serotype level. In the second approach, the spectra generated from virus infected cell cultures revealed the presence of some different peaks not overlap- ping those of uninfected cell cultures for all the reference virus infected cell cultures. The parameters for the creation of the Main Spectrum Profile (MSP) for each of the reference virus infected cell cultures were set on the basis of these peaks. The obtained MSP spectra were used to create a new respiratory viruses library in our Bruker Daltonics database in order to blind identify viruses isolated from biological samples aster a cell culture step. The spectra obtained by 58 additional cultured strains correctly match with the new database demonstrating its reliability. Discussion and Conclusions. In conclusion, this study could be considered a starting point for further evolutions of the developed system, since the differences observed comparing the spectra obtained from virus infected cell culture suggest the possibility to apply these approaches to the identification of other viruses including other picornavirus such as enterovirus and coxsackievirus and viruses responsible for respiratory infections, as well as to viral agents causing infections of other body sites.
An innovative application of MALDI-TOF MS in clinical virology / Calderaro, Adriana; Buttrini, Mirko; Montecchini, Sara; Motta, Federica; Rodighiero, Isabella; Arcangeletti, Maria Cristina; Chezzi, Carlo; DE CONTO, Flora. - (2018), p. 153. ((Intervento presentato al convegno 46° Congresso Nazionale della Società Italiana di Microbiologia tenutosi a Palermo nel 26-29 settembre2018.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2851578
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