Efficient purification of cell culture-derived classical swine fever virus by ultrafiltration and size-exclusion chromatography

Abstract

Large-scale production of cell culture-based classical swine fever virus (CSFV) vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium. Hence, we have developed an efficient method for purifying CSFV from cell-culture medium. Pure viral particles were obtained with two steps of tangential-flow filtration (TFF) and size-exclusion chromatography (SEC), and were compared with particles from ultracentrifugation by transmission electron microscopy (TEM), infectivity and recovery test, and real time fluorescent quantitative PCR (FQ-PCR). TFF concentrated the virus particles effectively with a retention rate of 98.5%, and 86.2% of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system. CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10^−4.25 TCID₅₀·mL⁻¹ to 3.0×10^−6.25 TCID₅₀·mL⁻¹, but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone. In addition, pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination. Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV (P<0.05). The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.

Keywords

classical swine fever virus / virus purification / tangential-flow filtration / size-exclusion chromatography

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Acknowledgements

This research was supported by the Special Fund for Agro-scientific Research in the Public Interest (201203039) and China Agriculture Research System (CARS-36).

Ruining Wang, Yubao Zhi, Junqing Guo, Qingmei Li, Li Wang, Jifei Yang, Qianyue Jin, Yinbiao Wang,Yanyan Yang, Guangxu Xing, Songlin Qiao, Mengmeng Zhao, Ruiguang Deng, and Gaiping Zhang declare that they have no conflict of interest or financial conflicts to disclose.

All applicable institutional and national guidelines for the care and use of virus were followed.

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg