Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system

Abstract

Background: Enterovirus 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 finding: In 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 >10(6) TCID(50)/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).

Conclusion: These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

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.

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.