Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

Abstract

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry.We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U S 2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.Bacterial artificial chromosomes (BAC) containing fulllength genomes of several herpesviruses have enabled the application of rapid mutagenesis strategies to identify functions of individual genes and determinants of pathogenicity (1). Marek's disease (MD) is a contagious lymphoproliferative disease of poultry caused by the highly oncogenic alphaherpesvirus MD virus (MDV). The application of BAC mutagenesis strategies to study MDV oncogenicity has been hampered by the nonavailability of BAC clones of oncogenic strains, as two of the previously reported BAC clones derived from attenuated MDV strains were unable to induce tumors (4, 7). The RB-1B strain of MDV isolated in the early 1980s is a highly oncogenic strain (5, 6) that consistently induces a high incidence of MD with rapid-onset tumors in visceral organs. We have previously shown that RB-1B infection in 1-week-old Rhode Island red chickens caused a 100% incidence of MD with tumors in visceral organs 6 to 7 weeks after infection (4). Taking advantage of this high oncogenic potential, we have chosen the RB-1B strain for the construction of BAC clones to identify oncogenic determinants of MDV.A clone-purified fourth-passage stock of the RB-1B strain of MDV (5), tested free of avian leukosis, reticuloendotheliosis, and chicken infectious anemia viruses, was used for the preparation of viral DNA for the construction of RB-1B BAC clones. Viral DNA was prepared from chicken embryo fibroblast (CEF) cultures by sodium dodecyl sulfate-proteinase K extraction (7). RB-1B BAC construction was carried out by insertion of the mini-F plasmid pHA1 into the U S 2 gene of MDV essentially as previously described (7). Briefly, secondary CEF cultures were cotransfected with RB-1B virus-infected genomic DNA and plasmid pDS-pHA1 (7) and passaged five or six times on primary CEF in medium containing 250 g of mycophenolic acid, 50 g of xanthine, and 100 g of hypoxanthine per ml at 4-day intervals. DNA from these cells was electroporated into Escherichia coli DH10B cells and plated on Luria-Bertani plates containing 30 g of chloramphenicol per ml. Transfection of high-molecular-weight extrachromosomal BAC DNA from three single colonies (designated pRB-1B-1, pRB-1B-2, and pRB-1B-5) into primary CEF produced MDVspecific plaques in about 4 to 5 days after transf...

Expression profiles of microRNAs encoded by the oncogenic Marek's disease virus reveal two distinct expression patterns in vivo during different phases of disease

“…Although whether or not REV-LTR insertion affects the in vivo expression of MDV-1-encoded miRNAs is unknown as yet, our research may provide meaningful clues for further evaluating their functions, especially for the twelve miRNAs highlighted in this study. Based on the successful establishment of strategies for mRNA target prediction and experimental identification (Kuhn et al, 2008;Alexiou et al, 2009;Bartel, 2009), and the application of bacterial artificial chromosomes in research on MD pathogenesis (Schumacher et al, 2000;Petherbridge et al, 2003Petherbridge et al, , 2004Sun et al, 2009Sun et al, , 2010, focusing on those miRNAs shown to be expressed in vivo in this study should reveal more about the biological functions of MDV-1-encoded miRNAs.…”

Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek’s disease virus

“…This opportunity has been possible because of the availability of the infectious BAC clone of the highly oncogenic RB-1B strain of MDV (19), which allows the application of mutagenesis techniques for the rapid generation of mutant viruses. Pathogenicity studies in susceptible inbred and outbred chickens clearly demonstrated that MEQ-CtBP interaction is crucial for the induction of lymphomas because the pRB-1B-Ct20 virus with mutation in the MEQ genes that abolished the interaction with CtBP was completely nononcogenic.…”

“…A DF-1 cell line derived from line 0 chicken embryonic fibroblasts (27) was used for immunoprecipitation and reporter assays. The very virulent RB-1B strain of MDV originally obtained from K. A. Schat (Cornell University, Ithaca, NY) and the BAC-derived pRB-1B5 viruses (19) were used in pathogenicity tests.…”

“…The availability of an infectious BAC clone (pRB-1B5) of the highly oncogenic RB-1B strain of MDV (19) provided us with the opportunity to use reverse genetics to construct mutant CtBP-interaction-defective viruses. We made use of the markerless replacement technique (20) in the pRB-1B5 clone to construct four MDV mutants (Fig.…”

Relationship Between Levels of Very Virulent MDV in Poultry Dust and in Feather Tips from Vaccinated Chickens

“…pCVI988-10 and Poulvac CVI988 both give 100% protection against mortality following infection with a vvMDV strain (28). Group 3 received pRB-1B-2, a BAC-cloned experimental virus which is derived from vvMDV strain RB-1B (31) but has genomic deletions that result in a loss of pathogenicity and oncogenicity (27). Group 4 received a bivalent vaccine, HVT+SB-1 (Marexine, Intervet).…”