Figure 1.
Purification of the EV71 virus by sucrose gradient zonal ultracentrifugation.
The concentrated EV71 harvest stock was separated into 25 fractions. A. The viral titer of each fraction was determined by a TCID50 assay. B. The EV71 antigens were detected by western blot using MAb979. P means the positive control that is the formalin-inactivated EV71/E59 vaccine bulk produced by roller bottle method.
Figure 2.
Photographs of EV71 viral particles as analyzed by transmission electron microscopy.
(A) Fraction 10 was empty and had a defective particle (E-particle) structure. (B) Fraction 16 was full and had a solid particle (F-particle) structure. The bar represents 50 nm.
Figure 3.
The viral antigen composition of the EV71 viral particle was analyzed by SDS-PAGE and western blot.
(A) Two different types of EV71 viral particles were analyzed on a NuPAGE 4–12% Birs-Tris Gel. (B) EV71 viral proteins were detected by the MAb979 antibody. (C) EV71 viral proteins were detected by the E1 antibody.
Table 1.
Summary of the predicted molecular weights (MW) of the EV71-E59 viral proteins and incomplete processed viral polypeptides [35].
Figure 4.
EV71 viral RNA content measured by RT-PCR.
The results of quantitative RT-PCR using primers specific for a 60 bp region of the EV71 VP1 gene are reported for the F-particle and E-particle by the blue line and red line, respectively.
Figure 5.
Western blot analyses of EV71 viral particles inactivated by formaldehyde.
The two different types of formalin-inactivated EV71 particles were separated on a NuPAGE 4–12% Bis-Tris Gel and analyzed by two monoclonal antibodies: (A) the MAb979 antibody and (B) the E1 antibody.
Table 2.
EV71 virus neutralization titers of mouse antisera generated against two different types of EV71-E59 viral particle fractions as measured by a TCID50 neutralization assay.