Reddy Sanjay M, Sun Aijun, Khan Owais A, Lee Lucy F, Lupiani Blanca
College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
Avian Dis. 2013 Jun;57(2 Suppl):469-73. doi: 10.1637/10444-110412-ResNote.1.
Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.
细菌人工染色体（BAC）载体最初是为便于大片段DNA的繁殖和操作而开发的。该技术后来被用于克隆大型DNA病毒的全长基因组，以研究病毒基因功能。马立克氏病病毒（MDV）是一种高度致癌的疱疹病毒，可在鸡体内迅速诱发T细胞淋巴瘤。根据该病毒在接种疫苗的鸡中引发疾病的能力，MDV毒株被分为不同的致病型，其中毒性最强的毒株属于超强毒（vv+）致病型。在此，我们报告了MDV超强毒株686（686-BAC）的BAC克隆构建。将从两个独立克隆中分离的DNA转染至鸭胚成纤维细胞后，可回收感染性病毒。致病性研究表明，BAC衍生的686病毒比MDV的vv毒株Md5毒性更强。通过两步红介导诱变过程，从MDV基因组中删除了病毒白细胞介素8（vIL-8）的两个拷贝，表明686-BAC适合诱变技术。从MDV的vv+毒株产生BAC克隆是朝着理解MDV发病机制的分子基础迈出的重要一步。
