An 18to 20-nm virus particle was isolated from the Olson strain of quail bronchitis, an avian adenovirus. On density gradient separation the small virions were primarily found at densities of 1.39 and 1.42 g/cm3. The majority of the infectious particles were at the heavier density. The virus had a hexagonal outline and contained single-stranded deoxyribonucleic acid. It was resistant to heating at 56 C for more than an hour and was not inactivated by treatment with chloroform or low pH. Purified virus did not agglutinate erythrocytes of various avian and mammalian species. Replication of the small particles occurred either in chicken embryos or in cultures of embryo kidney cells coinfected with an adenovirus helper. Antigenically the virus was distinct from the adeno-associated viruses types 1, 2, 3, and 4. The virus is the avian equivalent of the adeno-associated viruses of primates and lower animals. The adeno-associated viruses are defective and have been found in association with the
Avian adenoviruses were isolated from 56 of 106 fecal and rectal tissue samples taken from apparently healthy young chickens on 4 farms. Only one isolation was made from the 37 samples from 3- and 4-week-old chicks, while the isolation frequency was 64-100% in groups 5 weeks old and older. The 56 adenovirus isolates were classified into 6 serotypes. Vurses of 3 or 4 types were found on each farm. Avian adeno-associated virus CF antigens were found, using chicken embryos coinfected with an adenovirus helper, in 2 of the 38 adenovirus isolated studied.
Chicken-embryo-lethal-orphan (CELO) virus, Phelps strain, agglutinated erythrocytes at 37 C. The hemagglutinating activity, which is a function of complete and incomplete virus particles, was sensitive to heat but not to pH. The soluble components of the virus were similar in sedimentation characteristics to those obtained from human adenovirus type 1. The effects of chemical and physical agents on CELO hemagglutinin, CELO infectivity, and red-cell receptors suggested that the last were protein in nature and that cell-virus attachment was mediated by amino groups on the virion. The attachment of virus to red blood cells via the penton projection was demonstrated by electron microscopy.
Eleven avian adenovirus strains were tested for the presence of avian adeno-associated viruses (AAAV). Six strains contained AAAV. Electron microscopy using rabbit anti-AAAV serum was useful in detecting the satellite virus. The AAAV previously isolated from quail bronchitis virus was related to each of the six new isolates by immunoagglutination, complement fixation, immunodiffusion, and neutralization tests.
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...
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