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

Overview

Journal Food Environ Virol

Publisher Springer

Specialties Environmental Health
Microbiology
Nutritional Sciences

Date 2013 Aug 13

PMID 23934702

Citations 6

Authors

Kata Farkas

Liping Pang

Susan Lin

Wendy Williamson

Richard Easingwood

Rayleen Fredericks

Mohamed A Jaffer

Arvind Varsani

Affiliations

Soon will be listed here.

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 × 10 particles and we note that estimates by qNano are similarly skewed (1.36 × 10 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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