title: Immunological and biochemical characterization of coxsackie virus A16 viral particles.
abstract: BACKGROUND
  Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.
  
  
  RESULTS
  In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10(6) the tissue culture's infectious dose (TCID(50)) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10(-5) was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24-28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176-190.
  
  
  CONCLUSIONS
  These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.
authors.affiliation: Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan. pelechong@nhri.org.tw
authors.forename: Pele
authors.initials: P
authors.lastname: Chong
authors.forename: Meng-Shin
authors.initials: MS
authors.lastname: Guo
authors.forename: Fion Hsiao-Yu
authors.initials: FH
authors.lastname: Lin
authors.forename: Kuang-Nan
authors.initials: KN
authors.lastname: Hsiao
authors.forename: Shu-Yang
authors.initials: SY
authors.lastname: Weng
authors.forename: Ai-Hsiang
authors.initials: AH
authors.lastname: Chou
authors.forename: Jen-Ren
authors.initials: JR
authors.lastname: Wang
authors.forename: Shih-Yang
authors.initials: SY
authors.lastname: Hsieh
authors.forename: Ih-Jen
authors.initials: IJ
authors.lastname: Su
authors.forename: Chia-Chyi
authors.initials: CC
authors.lastname: Liu
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000276:immunology; D019149:Bioreactors; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D029821:Enterovirus A, Human / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure; D000939:Epitopes / Q000276:immunology; D006232:Hand, Foot and Mouth Disease / Q000517:prevention & control; D006801:Humans; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009500:Neutralization Tests; D011817:Rabbits; D014709:Vero Cells; D014771:Virion / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification* / Q000648:ultrastructure
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D000939:Epitopes