Purification of Infectious EV71 Particles

It is well known that two forms of EV71 viral particles, full and empty particles, existed during propagation in cells [3] (link), [26] (link). Based on historical poliovirus studies, the full particles are infectious and immunogenic but the empty particles are not [27] (link). Therefore, we purified EV71 infectious (full) particle of the reference viruses for rabbit immunizations. The EV71 culture supernatant was concentrated 10-fold with a Amicon 100K centrifugal filter (Millipore). The crude virus concentrate was loaded onto a 15–65% continuous sucrose gradient and centrifuged at 28000 rpm for 4 hr. Fractions (2 mL per fraction) were collected and the viral titer and protein concentration of each fraction were determined by TCID₅₀ and BCA assays (Thermo Scientific), respectively. Fractions with high infectious virus titers in 32–38% sucrose concentration were merged and concentrated by diafiltration using Amicon 100K centrifugal filter and centrifugation at 3500 g. The purified EV71 viruses were further verified by using Western blot and electron microscopy analysis.