Meehan B M, Todd D, Creelan J L, Connor T J, McNulty M S
Department of Veterinary Science, The Queen's University of Belfast, United Kingdom.
J Virol. 1997 Nov;71(11):8362-7. doi: 10.1128/JVI.71.11.8362-8367.1997.
Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted 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. McNulty, 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 region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia 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 nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.
鸡贫血病毒（CAV）的Cux - 1分离株在细胞培养中传代173次后进行分子克隆，得到了一株减毒株，命名为克隆分离株10，该毒株在幼雏中传代10次后恢复了毒力（D. 托德、T. J. 康纳、V. M. 卡尔弗特、J. L. 克里兰、B. M. 米汉和M. S. 麦克纳尔蒂，《禽病学》24:171 - 187，1995年）。减毒的克隆分离株10与分子克隆的致病性Cux - 1分离株不同，在于它在CAV基因组的非转录区域有一个21个核苷酸的插入，并且在整个基因组中有17个单个核苷酸替换。与其他已发表的CAV序列进行比较分析表明，克隆分离株10在9个核苷酸位置和5个氨基酸位置是独特的。通过评估两组互补嵌合病毒的致病性，研究了克隆分离株10表现出的减毒分子基础。这些组是通过用嵌合双链复制型（RF）DNA等效物转染产生的，这些等效物包含来自克隆分离株10和致病性克隆Cux - 1分离株的DNA序列。嵌合RF的构建利用了独特的EcoRI、PstI和BamHI限制性酶切位点的出现，这使得它们各自的环状CAV RF可以作为0.58、0.93和0.71 kbp的三个限制性片段进行操作。用嵌合和亲本克隆分离株感染1日龄雏鸡后，检查14日龄无特定病原体雏鸡的贫血水平以及胸腺和骨髓的大体病理学，结果表明三个基因组区域中的每一个区域的核苷酸变化都对减毒有贡献。讨论了这一结果对减毒活CAV疫苗开发和使用的意义。
