Reconstituting turkey herpesvirus with bacterial artificial chromosome clones

Abstract:

Herpesvirus of turkey (HVT) is an alpherpesvirus and widely used as a live vaccine against Marek’s disease (MD) because of its antigenic relationship with Marek’s disease virus (MDV). [Objective] The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome (BAC). [Methods] Using the selection marker Eco-gpt (Xanthine-guanine phosphoribosyl transferase)(1.3 kb) and BAC vector pBeloBAC11(7.4 kb), we constructed the transfer plasmid pGAB-gpt-BAC11. Then, the transfer plasmid and HVT-infected cells’ total DNA were cotransfected into primary chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, we obtained purified recombinant viruses. Genomic DNA was extracted and electroporated into Escherichia coli DH10B competent cells. BAC clones were identified by restriction enzyme digestion and PCR analysis, and then tested for infectivity after transfection into CEFs using calcium phosphate. [Results] We obtained 25 BAC clones, and reconstituted recombinant viruses by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively. [Conclusion] In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome.