Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera

Li, Ji Lian; Cornman, R. Scott; Evans, Jay D.; Pettis, Jeffery S.; Zhao, Yan; Murphy, Charles; Peng, Wen Jun; Wu, Jie; Hamilton, Michele; Boncristiani, Humberto F.; Zhou, Liang; Hammond, John; Chen, Yan Ping

Virus purification and electron microscopy. Thirty worker bees were frozen in liquid nitrogen, ground to a fine powder, and homogenized in 10-ml extraction buffer (0.1 M potassium phosphate buffer [pH 7.5], 0.2% diethyldithiocarbamate, 1/5 volume of diethyl ether). The mixture was emulsified with 5 ml carbon tetrachloride and centrifuged at 5,000 ϫ g at 4°C for 30 min to remove tissue debris. Supernatant containing viruses was centrifuged once more at 5,000 ϫ g at 4°C for 30 min and then filtered through a 45-m filter to remove small tissue debris. The filtrate was then centrifuged at 10,187 ϫ g for 6 h at 4°C to pellet the viral particles. The pellet was resuspended in 2 ml of 0.2 M phosphate-buffered saline (PBS) buffer. A 15-l portion of viral solution was examined for the presence of virus particles in an electron microscope. The rest of the viral solution was saved for subsequent viral RNA isolation and cDNA library construction.