METHODS FOR VIRAL ISOLATION AND DNA EXTRACTION FOR A PENAEID SHRIMP BACULOVIRUS

Procedures for the purification of virions and nucleocapsids of Baculovirus penaei (BP) of penaeid shrimp and subsequent extraction of the viral nucleic acid are described. BP-infected hepatopancrata, from two species of shrimp from different geographical locations in the Americas, were removed and homogenized in a solution of TN buffer (0.01 M Tris-HCl, 0.10 M NaCl, pH 8.0). The homogenized mixture was strained through a 100-mesh screen to remove large pieces of tissue and centrifuged to concentrate the remaining material. The pellet was suspended in TN buffer and layered on to a handmade CsCl gradient. Fractions were collected according to the bands observed in the gradient, and the optical density at 254 nm was recorded for each fraction. The resultant data was tabulated and graphed. Additionally, each fraction was examined by transmission electron microscopy to determine relative numbers of viral particles present. Large amounts of virus consistently corresponded to a specific band in the gradient, which produced a peak when the spectrophometric data was graphed. Nucleic acid was then extracted from the purified viral particles. Removal of polysaccharides was accomplished with the addition of CTAB/NaCl. The BP DNA was visualized on an agarose gel with phage lambda DNA markers for size estimation, and a preliminary endonuclease digestion was performed using BamHI.