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A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications

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Abstract

This article describes a rapid method for purifying infectious rotavirus particles from cell culture for environmental research. The method is based on size-exclusion chromatography using TOSOH TSKgel® G5000PWXL-CP with a TSKgel® Size Exclusion G2500PWxl guard column, set up on an AKTA Explorer10. Four peaks were identified from the chromatogram and the corresponding fractions were collected and analysed by electron microscopy, 1-step quantitative reverse transcription polymerase chain reaction (RT-PCR) and qNano measurement. Infectivity potential of the recovered virus particles was determined using cell culture. Our analysis reveals that the first fraction contains majority of the intact triple-layered infectious virions while the other three fractions contain mixtures of empty capsids and intact infectious virions. Our results also indicate that there is a gross overestimation of rotaviruses in crude extracts due to encapsidated RNA in the order of 2.3 × 1011 particles and we note that estimates by qNano are similarly skewed (1.36 × 1013 particle) possibly due to empty capsids and cellular debris. In summary we present a method for purification (~12 h) of rotaviruses for a more robust and accurate quantification of virus size, surface charge and particle concentration in environmental contexts.

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Acknowledgments

This study was part of a Marsden Project funded by the Royal Society of New Zealand under Contract Number ESR-1001. Kata Farkas is funded by a PhD scholarship from the Project. We wish to thank Dr. Jeremie Langlet and Dr. Joanne Hewitt (Institute of Environmental Science and Research Ltd.) for their general discussions on virus purification.

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Authors and Affiliations

  1. Institute of Environmental Science & Research Ltd., PO Box 29-181, Christchurch, New Zealand

    Kata Farkas, Liping Pang, Susan Lin & Wendy Williamson

  2. School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand

    Kata Farkas, Wendy Williamson & Arvind Varsani

  3. Otago Centre for Electron Microscopy, University of Otago, PO Box 56, Dunedin, New Zealand

    Richard Easingwood

  4. Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand

    Rayleen Fredericks & Arvind Varsani

  5. Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, 7700, South Africa

    Mohamed A. Jaffer & Arvind Varsani

Authors
  1. Kata Farkas
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  2. Liping Pang
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  3. Susan Lin
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  4. Wendy Williamson
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  5. Richard Easingwood
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  6. Rayleen Fredericks
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  7. Mohamed A. Jaffer
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  8. Arvind Varsani
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Correspondence to Arvind Varsani.

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Farkas, K., Pang, L., Lin, S. et al. A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications. Food Environ Virol 5, 231–235 (2013). https://doi.org/10.1007/s12560-013-9122-4

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  • DOI: https://doi.org/10.1007/s12560-013-9122-4

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