Purification of Murine Gammaherpesvirus 68 With Use of Differential Centrifugation

The method for separation of viral particles in a concentrated form from the environment is called virus purification. Viruses are required to be purified for a range of studies in which it is necessary to distinguish the properties or structure of a virus from the host cells or culture media, including analysis of viral polypeptide structures and membrane glycoprotein function. Our objective was to purify murine gammaherpesvirus 68 (MHV-68, MuHV-4) using the centrifuge, equipment and other materials available in our laboratory. After infection of baby hamster kidney 21 (BHK-21) cells with MHV-68 with the multiplicity of infection (MI) of 0.01 and following virus multiplication, we repeatedly froze and thawed the cell culture to disrupt the cells and release the virus particles into the culture medium. We used low-speed centrifugation (3000 rpm at 4°C) to separate the viral particles from cell debris. Subsequently, we transferred the supernatant containing virus particles to a fresh centrifuge tube and centrifuged at a speed of 8000 rpm (8801 g) and 11,000 rpm (=16,639 g) and at 4°C. We tested different centrifugation durations of 2, 4, 6 and 8 hours. To evaluate the quality of the obtained purified MHV-68 virus by this method and compare it to purified MHV-68 sample acquired by conventional ultracentrifugation on sucrose cushion (30%, w/v), we used the SDS-PAGE separation method using a 4%–20% (w/v) and 6%–14% (w/v) gradient gel. We obtained the best results with 6-hour-long centrifugation at 11,000 rpm. In conclusion, we managed to optimise virus purification method using the equipment available in our laboratory and prepared purified MHV-68 virus in sufficient concentration for determination of MHV-68 virus proteins.

EVALUATION OF VIRAL PARTICLES YIELD AT DIFFERENT STAGES OF ADENO-ASSOCIATED VIRUS PURIFICATION

Here we present results of a comparison of two methods of purification of adeno-associated virus: iodixanol gradient ultracentrifugation and polyethylene glycol solution (PEG 8000) two-stage purification.