Construction Of Marek's Disease Virus Very Virulent Strain L-ZY As A Bacterial Artificial Chromosome

Marek's disease(MD) which is a lymphoproliferative disease of chickens,is of great concern for the poultry industry.In the absence of control measures,MD is capable of causing devastating losses in poultry flocks.MD is induced by Marek's disease virus or MDV.Since the 1950s,HVT,HVT and naturally non-oncogenic serotype 2 MDV strains,attenuated serotype 1 MDV were introduced validly in succession to control this disease.However,during the 1990s the very virulent plus (â…´â…´+) MDV strains which is capable of breaking through the barriers of immune protection resulted in a significant incidence of MD in flocks.MDV L-ZY strain is a very virulent virus strain which was isolated in the northeastern region of China in 2006.MDV L-ZY strain can result in 70%occurrence of immune chickens with MD,and has a close genetic relationship with several very virulent virus strain separated in China.The emergence of very virulent strains is a new challenge for the control of MD.Therefore,it is of great importance to study the disease mechanism of MD.MDV is strictly cell-associated virus,it is difficult to directly operate on the gene level for the virus.However,the development of a novel approach "bacterial artificial chromosome,BAC" can solve the problem.The aim of the study is to construct MDV L-ZY strain bacterial artificial chromosome.To construct the BAC transfer vector,two fragments on either side of the MDV L-ZY US2 gene,which acted as left- and right-side homologies,selective marker guanine phosphoribosyl transferase(gpt) cassette,two loxP sites and BAC vector pBeloBAC11 were inserted in pUC119 clone vector.The two loxP sites,the direction of which is the same,was located on both side of the BAC-gpt sequence. Chicken embryo fibroblasts(CEFs) were co-transfected with pUAB-gpt-BAC and MDV L-ZY DNA by calcium phosphate transfection agent.Incubation was continued until the cells showed obvious CPE.To enrich the medium for recombinant virus(rv-MDV L-ZY),the virus-containing cells were passaged in the selection medium for 6 rounds.The DNA extracted from recombinant virus-infected cells was electroporated into E.coli DH10B cells and BAC DNA was obtained.BAC DNA were identified by restriction enzyme digest,PCR and gel electrophoresis analyses.Finally,we obtained 21 BAC clones.DNA from the L-ZY BAC21 clone was transfected into CEFs,and recombinant virus was reconstituted.We cloned the complete genome of MDV L-ZY strain as an infectious bacterial artificial chromosome and named derived-MDV L-ZY21.In this study,we successfully constructed MDV serotype I very virulent L-ZY strain as an bacterial artificial chromosome and perfect MDV BAC reverse genetic system which can help the understanding of tumorigenicity and gene function of MDV.In addition,it opens the possibility to generate novel MDV-1 vaccines based on the BACs.