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

Kata Farkas; Liping Pang; Susan Lin; Wendy Williamson; Richard Easingwood; Rayleen Fredericks; Mohamed A. Jaffer; Arvind Varsani

doi:10.1007/s12560-013-9122-4

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

Kata Farkas, Liping Pang, Susan Lin, Wendy Williamson, Richard Easingwood, Rayleen Fredericks, Mohamed A. Jaffer, Arvind Varsani

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

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.

Original language	English (US)
Pages (from-to)	231-235
Number of pages	5
Journal	Food and Environmental Virology
Volume	5
Issue number	4
DOIs	https://doi.org/10.1007/s12560-013-9122-4
State	Published - Dec 2013
Externally published	Yes

Keywords

Characterisation
Purification
Quantification
Rotavirus
Size-exclusion chromatography

ASJC Scopus subject areas

Epidemiology
Food Science
Health, Toxicology and Mutagenesis
Virology

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10.1007/s12560-013-9122-4

Cite this

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

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{\textregistered} G5000PWXL-CP with a TSKgel{\textregistered} 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.",

keywords = "Characterisation, Purification, Quantification, Rotavirus, Size-exclusion chromatography",

author = "Kata Farkas and Liping Pang and Susan Lin and Wendy Williamson and Richard Easingwood and Rayleen Fredericks and Jaffer, {Mohamed A.} and Arvind Varsani",

note = "Funding Information: 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.",

year = "2013",

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doi = "10.1007/s12560-013-9122-4",

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T1 - A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications

AU - Farkas, Kata

AU - Pang, Liping

AU - Lin, Susan

AU - Williamson, Wendy

AU - Easingwood, Richard

AU - Fredericks, Rayleen

AU - Jaffer, Mohamed A.

AU - Varsani, Arvind

N1 - Funding Information: 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.

PY - 2013/12

Y1 - 2013/12

N2 - 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.

AB - 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.

KW - Characterisation

KW - Purification

KW - Quantification

KW - Rotavirus

KW - Size-exclusion chromatography

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

Abstract

Keywords

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