Isolation of the nucleic acid of Newcastle disease virus (NDV).

Abstract

particle as multiple pieces in certain cases.5 6 Recently, in this laboratory, the intact RNA was isolated from the mixture of Rous sarcoma virus (RSV) and its helper virus RAV which belong to the avian leukosis complex of viruses.7 This result suggested that similar methods be used with a member of the myxovirus group. This paper describes the purification of NDV and the isolation of a 57S2o RNA, one molecule of which probably accounts for the total RNA per virus particle. While this study was in progress a brief report appeared on the isolation of a 45-50S RNA from NDV.8 Materials and Methods.-The plaque-forming L-Kansas 48 strain of NDV was used in all experiments. Ten-day-old eggs were inoculated in the allantoic cavity with 1-3 X 102 PFU of virus. After incubation at 40?C for 40-44 hr, the eggs were chilled and the chorioallantoic fluid was harvested. To remove cell debris the fluid was centrifuged at 4000 rpm for 10 min in a Servall GSA rotor. The supernatant was then kept overnight at 4?C. Virus purification: The virus was purified in three steps, all carried out in the cold (0-4?C). The following procedure was used to purify 800-1800 ml of allantoic fluid. First step: Centrifugation to a density interface: Twenty-eight ml of allantoic fluid was layered on 5 ml of a sucrose-RbCl solution and centrifuged in the Spinco 30 rotor for I hr at 30,000 rpm. The sucrose-RbCl solution contained 60% sucrose, 15% RbCl, 0.01 M Tris hydroxymethylaminomethane HCl (Tris - HCl), pH 7.3, and 0.001 M versene (EDTA); its density was 1.34gm per ml. After centrifugation a visible band of virus was seen at about density 1.22 gm per ml in the steep density gradient between the sucrose-RbCl solution and the allantoic fluid. A second band of nonviral material appeared 3-5 mm above the virus zone at about density 1.04 gm per ml. The tube was then punctured at the bottom, the virus band collected, and the upper band of foreign material discarded. The virus solution was diluted with an equal volume of buffer, 0.01 M Tris HCI, pH 7.3, 0.001 M EDTA and 1/4 vol of medium containing 4% calf serum.9 Second step: Ammonium sulfate precipitation: An equal volume of saturated ammonium sulfate, previously neutralized with Tris HCI buffer, was added dropwise to the virus solution. The precipitate was collected by centrifugation, and the supernatant containing about 1% of the starting infectivity was discarded. Third step: Sucrose-D2O gradient: The precipitate was redissolved in buffer containing 0.01 M Tris HCl, pH 7.3, 0.1 M NaCl, and 0.001 M EDTA to make a final volume between 4 and 9 ml depending on the amount of virus present to ensure that the solution density was less than 1.07 gm per ml. Up to 4.5 ml of the virus solution was then layered on top of an 18-ml linear gradient of sucrose in heavy water and then overlaid with mineral oil to fill the tube and centrifuged in a Spinco SW25 rotor at 25,000 rpm for 6 hr. The gradient was made, using 65% sucrose in D20 (density 1.32 gm per ml) and an equal volume of that solution diluted 4-fold with H20. Both solutions and the final gradient contained 0.01 M Tris-HC1, pH 7.3, 0.05 M NaCl, and 0.001 M EDTA. The virus band could be seen at about the middle of the tube. A second band of nonviral ma