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. 2012;7(11):e49973.
doi: 10.1371/journal.pone.0049973. Epub 2012 Nov 30.

Immunological and biochemical characterization of coxsackie virus A16 viral particles

Pele Chong  1 , Meng-Shin Guo, Fion Hsiao-Yu Lin, Kuang-Nan Hsiao, Shu-Yang Weng, Ai-Hsiang Chou, Jen-Ren Wang, Shih-Yang Hsieh, Ih-Jen Su, Chia-Chyi Liu
Affiliations

Immunological and biochemical characterization of coxsackie virus A16 viral particles

Pele Chong et al. PLoS One. 2012.

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.

Principal finding: 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.

Conclusion: These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The up-stream process for CVA16 virus production.
The consistency of 3 lots of CVA16 virus produced from Vero cells grown in VP-SFM medium (run #1 to 4) in a 5 L microcarrier bioreactor system was monitored. Virus titer was detected every day by TCID50 for 14 days.
Figure 2
Figure 2. CVA16 purification using sucrose gradient zonal ultracentrifugation.
The concentrated CVA16 harvest stock was separated into 25 fractions. (A) The viral antigens of each fraction were analyzed by SDS-PAGE and then sliver-stained. (B) EV71 antigens were detected by Western blot using MAb979.
Figure 3
Figure 3. Transmission electron microscopy imaging of formalin-inactivated CVA16 viral particles.
(A) Fraction 10 was empty and had a defective particle (P-particle) structure. (B) Fraction 16 was full and had a solid particle (R-particle) structure.
Figure 4
Figure 4. SDS-PAGE analysis of viral antigen composition of CVA16 and EV71 viral particles.
CVA16 P-particles (lane 1), CVA16 R-particles (lane 2), EV71 P-particles (lane 3), and EV71 R-particles (lane 4) were analyzed on a NuPAGE 4–12% Birs-Tris Gel.
Figure 5
Figure 5. CVA16 viral RNA content measured by RT-PCR.
The results of quantitative RT-PCR using primers specific for a 60 bp region of the CVA16 VP2 gene are reported for the P-particle and R-particle by the blue line and red line, respectively.
Figure 6
Figure 6. Western blot analysis of reactivity of enterovirus antigens with mouse anti-sera raised against formalin-inactivated CVA16 particles.
R-particles derived from CVA16 and EV71 were separated on a NuPAGE 4–12% Bis-Tris Gel and analyzed using different antibodies: (A) anti-sera generated from formalin-inactivated CVA16 P-particles; (B) anti-sera generated from formalin-inactivated CVA16 R-particles; and (C) EV71-specific monoclonal antibody MAb N1.
Figure 7
Figure 7. Immunodominant B-cell linear epitope mapping against mouse anti-sera raised against CVA16 formalin-inactivated P-particles (left panel) and formalin-inactivated R-particles (right panel).
Peptide-ELISA format was used in covering all VP1, VP2, VP3 and VP4 sequences of CVA16.
See this image and copyright information in PMC

References

    1. McMinn PC (2002) An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26: 91–107. - PubMed
    1. Schmidt NJ, Lennette EH, Ho HH (1974) An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 129: 304–9. - PubMed
    1. Ho M, Chen ER, Hsu KH, Wu SJT, Chen KT, et al. (1999) The Taiwan Enterovirus Epidemic Working Group. An epidemic of enterovirus 71 infection in Taiwan. N Engl J Med 341: 929–935. - PubMed
    1. Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, et al. (2009) Reemergence of enterovirus 71 in 2008 in Taiwan: Dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Mincrobiol 47: 3653–3662. - PMC - PubMed
    1. Lin KH, Hwang KP, Ke GM, Wang CF, Ke LY, et al. (2006) Evolution of EV71 genogroup in Taiwan from 1998 to 2005: an emerging of subgenogroup C4 of EV71. J Med Virol 78: 254–262. - PubMed

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