Isolation of adenovirus-associated viruses from man.

The adenovirus-associated viruses (AAV) are a recently described group of DNA viruses that are unique in that they require an unrelated virus (adenovirus) for their replication.1-5 They have heretofore been described only as contaminants of laboratory stocks of human and simian adenoviruses; their natural history is un-known. Virus isolation procedure: For AAV and adenovirus isolation, 0.1 ml of each anal or throat specimen was inoculated into four HEK tubes, two of which had been preinfected for 1 hr with Ad. 12 at a virus/cell multiplicity of 1. These Ad. 12-preinfected tubes were frozen at complete adenovirus cytopathic effect (CPE), which occurred at 5 days. One-tenth ml of the fluid was then passed to two fresh HEK tubes; these were also frozen at complete CPE. Both first and second passage fluids were tested for AAV CF antigen by reaction against guinea pig reference antisera2 to AAV 1, 2, and 3. The other two HEK tubes, inoculated with clinical specimens without helper, were observed for adenovirus CPE for at least 21 days. Positive fluids were passed, and both first and second passage fluids were tested for AAV CF antigen. Adenovirus isolates were typed by neutralization tests as previously described.10 All AAV-positive specimens were retested; in 15 of 18 instances AAV of the same type was reisolated. The 3 instances where AAV could not be reisolated are not considered as isolates here. All isolation work was done in a closed laboratory cubicle in which no other adenovirus or AAV experimentation was performed. However, because of the remarkable stability of AAV, there is a high risk of laboratory contamination of tissue cultures and virus pools. Therefore, special control measures were included in all isolation experiments. Twenty to fifty per cent of cell cultures in each test received Ad. 12 alone; as with the tubes inoculated with clinical specimens, each Ad. 12-inoculated control tube was passed to a fresh HEK tube, which was assayed for AAV CF antigen. Also, representative Ad. 12 control tubes from each experiment were passed a total of six times and assayed for AAV at each passage level. These controls, consisting of 152 tubes re-ceiving adenovirus alone, were all negative for AAV. Thus, the three possible sources of AAV contamination-laboratory environment, HEK cells, and adenovirus helper-were effectively ruled out as spurious sources of isolations. Typing of AA V isolates: Most of the serologic characterization of AAV strains has been performed in CF, and the four known laboratory serotypes of AAV have been established primarily on this basis.2' The guinea pig sera used in this report were prepared from the reference strains of AAV, and were type-specific in CF, neutralization, and FA tests, except for low-level cross-reactions between AAV 2 and 3.12 The isolates reported here were classified as to serotype primarily by their reactivity in CF; they were further characterized using the following AAV neutralization test. Two-tenths ml of a 1: 3 dilution of the second passage fluid was heated at 560C for 15 min (to eliminate the infectious Ad. 12) and then was reacted for 45 min at room temperature with 0.2 ml of various dilutions of control and AAV type-specific guinea pig neutralizing antibody. The reaction mixtures were inoculated onto HEK cells that had been preinfected for 4 hr with Ad. 7 at a virus/cell multiplicity of 3. These tubes were frozen at complete CPE and examined for the presence of AAV in CF.