MOLECULAR-CLONING OF AN ATTENUATED CHICKEN ANEMIA VIRUS ISOLATE FOLLOWING REPEATED CELL-CULTURE PASSAGE

The pathogenicity of the Cux-1 isolate of chicken anaemia virus (CAV) was substantially reduced following large numbers (50 to 173) of passa ges in MDCC-MSB1 cells. Restriction endonuclease analysis of polymeras e chain reaction (PCR)-amplified DNAs and recombinant plasmids contain ing DNA inserts specified by CAV that had been passaged 173 times, ind icated that the population of high-passage virus was genetically diver se. A 21-base pair (bp) insertion, containing a 19-bp sequence identic al to four repeated sequences that are located in the putative non-cod ing region of the genome was shown to have become established in the v irus population by passage number 30. Individual virus isolates that w ere selected from the high-passage virus population using recombinant DNA cloning and transfection methodologies varied in their pathogenici ties. One cloned virus isolate, designated number 10, produced virtual ly no anaemia and substantially reduced levels of aplasia of the bone marrow and thymus atrophy. The pathogenicity of this isolate was resto red following 10 passages in young chicks.