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 micro- scopy (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 1.010 -4.25 TCID50$mL -1 to 3.010 -6.25 TCID50$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 immu- nized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultra- centrifugation-purified CSFV (P<0.05). The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.
Highlights
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and often fatal swine disease
Purification of CSFV has routinely been achieved by cesium chloride or sucrose density gradient ultracentrifugation[5,6], yet it was reported that the harsh force of ultracentrifugation might cause damage to viruses, especially to enveloped viruses[7], and bench scale gradient ultracentrifugation is unsuitable for larger scale processes[8]
An approximately 25-fold concentration of virus particles was obtained with a retention about 98.5% (Fig. 2a), indicating effective retention of virus particles
Summary
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and often fatal swine disease. It is an enveloped, singlestranded RNA virus with a genome of 12.3 kb and belongs to the genus pestivirus, the family Flaviviridae together with bovine viral diarrhea virus (BVDV), and border disease virus[1]. Large-scale production of cell culture-based CSFV vaccine is hampered because of the adverse reactions caused by the contaminants from host cell and cell culture medium. The development of an effective method for purifying CSFV virus from cell culture during vaccine manufacturing remains a research priority.
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