INVESTIGATION OF THE ATTENUATION EXHIBITED BY A MOLECULARLY CLONED CHICKEN ANEMIA VIRUS ISOLATE BY UTILIZING A CHIMERIC VIRUS APPROACH

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the iso lation of an attenuated strain, designated cloned isolate 10, which re verted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNu lty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed regio n of the CAV genome and 17 individual nucleotide substitutions dispers ed throughout the genome, Comparative analyses with other published CA V sequences indicated that cloned isolate 10 was unique at nine nucleo tide positions and at five amino acid positions, The molecular basis o f the attenuation exhibited by cloned isolate 10 was investigated by e valuating the pathogenicities of two sets of complementary chimeric vi ruses, These sets were produced by transfection with chimeric double-s tranded replicative-form (RF) DNA equivalents that contained DNA seque nces derived from cloned isolate 10 and the pathogenic cloned Cux-1 is olate, The construction of the chimeric RFs exploited the occurrence o f unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fr agments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anem ia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day old chicks with the chimeric and cloned parental isolates indicated that nucleoti de changes in each of the three genomic regions contributed towards at tenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.