Studies on an Oncogenic Avian Adenovirus (CELO) I. Biophysical Characterization

Abstract

The oncogenic activity of chick-embryolethal-orphan (CELO) virus has been described by various authors (1-4). Since CELO is commonly found in chicken eggs (5) and can transform both hamster (6, 7) and human (8) cells in vitro, its possible implication in human neoplasia warrants further study. In addition, some avian adenoviruses differ both immunologically (9) and in heat stability (10) from the adenoviruses in general ( 11). Therefore, we have attempted to determine whether the biophysical properties of CELO virus distinguish it from other members of the adenovirus group.Materials and Methods. Virus. The Phelps strain of CELO virus (5) was used. Stocks were produced by inoculating lo6 PFU/O.l ml of virus into the allantoic cavity of 10-day-old embryonated CELO-free eggs (Spafas, Inc., Norwich, Conn.). The eggs were incubated at 37' for 3 days. Before harvesting, the eggs were chilled overnight at 4'; the allantoic fluids were pooled and frozen at -20'. The virus pools used in these studies contained approximately lo9 PFU/ml. Plaque assay. This technique was described (12). Plaques were counted after incubation for 11 days.Complement-fixation tests. Our adaptation of the micro-complement-fixation (CF) test (13) was used. Fractions from density gradients were diluted 1 : 10 with Hanks' balanced salt solution (HBSS) and frozen at-20' until tested. Antiserum to CELO (14) was produced in adult LSH/LAK hamsters (15) by inoculation of density gradient purified virus. Antigens were detected using 4-8 units of antiserum.Human adenovirus group and control antigens, as well as the human reference serum, were purchased from Microbiological Associates, Bethesda, Maryland. lMicrohemaggZutination ( H A ) test.Rat erythrocytes (Microbiological Assoc.) were washed 3 times with phosphate buffered saline (PBS), pH 7.2. The 10-drop fractions from density gradient experiments were diluted with 0.1 ml of PBS. Twofold dilutions were prepared in 0.05-ml aliquots in microtiter TJ plates (Cooke Engineering Co.). The diluent contained 1 % normal rabbit serum.One drop (0.025 ml) of 1% (v/v) rat erythrocytes was added to each dilution. Plates were incubated at 37' until the cells in the control wells settled. Results. Purification of CELO virus.In preliminary experiments to visualize CELO virus by electron microscopy, we found that clarification of the allantoic fluid by light centrifugation (SOOg) removed most of the virus particles. Since titration of the virus consistently showed a titer of lo9 PFU/ml, it was suspected that much of the virus was embedded in debris present in the allantoic fluid. Previous studies by Black et aH. (16) indicated that SV40, also closely associated with cellular material, could be purified by treatment with sodium deoxycholate and trypsin. A combination of the methods of Black et al. (16) for SV40 and Green and Pina (17) for adenovirus was used in the purification of CELO virus. The allantoic fluid was treated with a final concentration of 1 % sodium deoxycholate and 0.01% trypsin at 37' for 30 min. The flui...