Abstract
BackgroundEnterovirus 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.MethodsCIM® 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.ResultsHighly 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%.ConclusionsEV-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.
Highlights
Enterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five
Sample preparation and concentration of virus One litre of cell culture supernatant containing EV-71 virus collected from T175 cm2 tissue culture flasks was used for purification
Concentration by polyethylene glycol (PEG) 8000 yielded a mixture of large amounts of viruses and other contaminant proteins from the Dulbecco’s Modified Eagle Medium (DMEM) and RD cells used for growing the virus
Summary
Enterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. Enterovirus 71, a close relative of polioviruses, was first isolated in California, USA in 1969 [1] Since it has become a major public health issue across Asia-Pacific region causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five [2]. The most effective way to control the disease caused by EV-71 is by vaccination and arises the need for the development of new Viruses possess various distinct characteristics some of which are: the number and distribution of positive or negative charges, distribution of aliphatic and aromatic hydrophobic residues and their size These virus characteristics can be utilized to fractionate them from other molecules [8]. The combination of PEG precipitation and monolithic chromatography was used for the purification of mycobacteriophage D29 [12]
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