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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS PATHOGENICITY, TRANSMISSION, AND EVOLUTION

Location: Avian Disease and Oncology Laboratory

Title: Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

Authors
item Reddy, Sanjay -
item Sun, Aijun -
item Khan, Owais -
item Lee, Lucy
item Lupiani, Blanca -

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 18, 2012
Publication Date: January 15, 2013
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.

Last Modified: 11/26/2014
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