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 passages in MDCC-MSB1 cells. Restriction endonuclease analysis of polymerase chain reaction (PCR)-amplified DNAs and recombinant plasmids containing DNA inserts specified by CAV that had been passaged 173 times, indicated that the population of high-passage virus was genetically diverse. A 21-base pair (bp) insertion, containing a 19-bp sequence identical to four repeated sequences that are located in the putative non-coding region of the genome was shown to have become established in the virus population by passage number 30. Individual virus isolates that were selected from the high-passage virus population using recombinant DNA cloning and transfection methodologies varied in their pathogenicities. One cloned virus isolate, designated number 10, produced virtually no anaemia and substantially reduced levels of aplasia of the bone marrow and thymus atrophy. The pathogenicity of this isolate was restored following 10 passages in young chicks.