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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Avian Disease and Oncology Research » Research » Publications at this Location » Publication #289325
Title: Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

Author
item REDDY, SANJAY - Texas A&M University
item SUN, AIJUN - Texas A&M University
item KHAN, OWAIS - Texas A&M University
item Lee, Lucy
item LUPIANI, BLANCA - Texas A&M University

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2012
Publication Date: 1/15/2013
Publication URL: http://handle.nal.usda.gov/10113/56837
Citation: Reddy, S.M., Sun, A., Khan, O.K., Lee, L.F., Lupiani, B. 2013. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome. Avian Diseases. 57(2):469-473.

Interpretive Summary: Mark’s disease (MD), a virus-induced cancer-like disease of chickens, is a major disease problem in commercial poultry. The objective of this work was to clone a very virulent plus strain of MD virus (MDV), 686, into a bacterial artificial chromosome (BAC) vectors in order to enable studies of viral gene function through manipulation of its genes. We have successfully constructed several BAC clones of 686 (686-BAC). Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than strain Md5, a very virulent strain of MDV. Using a molecular technique know as two-step red mediated mutagenesis process, both copies of a viral gene termed interleukin 8 (v-IL8) were deleted from the MDV genome, showing that 686-BACs were amenable to molecular manipulation (mutagenesis techniques). This is a first report of a generation of BAC clones from a vv+ strain of MDV and is a significant step towards understanding molecular basis of MDV pathogenesis.

Technical Abstract: 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 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 very virulent strain of MDV. Using a two-step red mediated mutagenesis process, both copies of viral interleukin 8 (v-IL8) 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 towards understanding molecular basis of MDV pathogenesis.