Abstract
BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).ConclusionThese results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.
Highlights
Enterovirus 71 (EV71) virus infection has recently caused outbreaks of hand-foot-and-mouth disease (HFMD) associated with severe neurological disease in young children [1,2] and has become a serious public health problem in the Asia-Pacific region [1,3,4,5,6,7,8] EV71 virus is a non-enveloped, single positive-stranded RNA virus of the family Picornaviridae with a genome size of approximately 7.4 kb
We describe the purification and biophysical characterization of EV71 viral particles that were produced from Vero cells grown in a serum-free microcarrier bioreactor system
The viral titers were determined by a TCID50 assay and found to reach 106/mL at 6 days post infection (DPI); there was no increase in viral titers in the 7th and 8th DPI
Summary
Enterovirus 71 (EV71) virus infection has recently caused outbreaks of hand-foot-and-mouth disease (HFMD) associated with severe neurological disease in young children [1,2] and has become a serious public health problem in the Asia-Pacific region [1,3,4,5,6,7,8] EV71 virus is a non-enveloped, single positive-stranded RNA virus of the family Picornaviridae with a genome size of approximately 7.4 kb. In previous murine immunogenicity studies, the formalin-inactivated EV71 virus produced from Vero cells elicited a more effective immune response than recombinant VP1 protein or DNA vector vaccines [17,18]. These results are difficult to compare because the standard production methods, standard virus neutralization assays and methods used to quantify the critical viral antigens are not available. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225)
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