A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications
Farkas K, Pang L, Lin S, Williamson W, Easingwood R, Fredericks R, Jaffer MA, Varsani A.
Food Environ Virol. 2013 Aug 11.
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.View Full Article