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Open Access Highly Accessed Methodology

Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

Ashok Raj Kattur Venkatachalam1, Milene Szyporta1, Tanja Kristin Kiener1, Premanand Balraj1 and Jimmy Kwang12*

Author Affiliations

1 Animal Health Biotechnology, Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore

2 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore

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Virology Journal 2014, 11:99  doi:10.1186/1743-422X-11-99

Published: 27 May 2014

Abstract

Background

Enterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials.

Methods

CIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer.

Results

Highly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%.

Conclusions

EV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.

Keywords:
Enterovirus 71; PEG precipitation; DEAE monolithic column