A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications.
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 × 1011particles and we note that estimates by qNano are similarly skewed (1.36 × 1013particle) 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.
Institute of Environmental Science & Research Ltd., PO Box 29-181, Christchurch, New Zealand.
Kata
K
Farkas
Liping
L
Pang
Susan
S
Lin
Wendy
W
Williamson
Richard
R
Easingwood
Rayleen
R
Fredericks
Mohamed A
MA
Jaffer
Arvind
A
Varsani