Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2007
Publication Date: 12/31/2007
Citation: Lee, L.F., Silva, R.F., Cui, X., Zhang, H., Heidari, M., Reddy, S. 2007. Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes. Avian Diseases. 51:851-857. Interpretive Summary: Marek's disease (MD), a virus-induced cancer-like disease of chickens, is considered as a major disease problem in commercial poultry. Vaccination has dramatically reduced the incidence of the disease, but very little is known about the basic mechanisms involved in the induction of disease. The objective of this research was to identify genes (hereditary elements) that control important virus functions such as replication and induction of tumor. We have used special (cosmid) technology to knockout a unique MDV gene (LORF11) and generated a mutant virus, rMd5/dLORF11. We characterized this mutant virus by studying its ability to replicate in chickens and found LORF11 gene is critical for viral replication. In addition, this mutant virus is also attenuated and did not induced tumor. This is a first report on finding an MDV gene that is essential for replication in chickens and tumor induction. This important new information will undoubtedly help scientists in academia and industry to understand gene function and designing better strategy to control MD.
Technical Abstract: DNA sequence data of the GA and Md5 strains of Marek's disease virus (MDV) revealed a large open reading frame (LORF11) located in the unique long region of MDV genome comprising 2711 nucleotides in length encoding a protein of 903 amino acids. Sequence comparison between MDV serotypes revealed that all three serotypes contained LORF11 homologs. No sequence homology was found with any other herpesviruses and therefore it is a unique MDV gene. The MDV-specific LORF11 gene was deleted from cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method (RARE). The recombinant cosmid, A6(dLORF11), was transfected into DEF cells in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. The recombinant rMd5(dLORF11) plaques were evident 12-13 days after transfection. The genomic structure was verified by PCR amplification of DEF cells infected with rMd5(dLORF11) viruses confirmed the deletion of a 2.57-Kb fragment and resulted in a 296-bp fragment. Restriction enzyme (RE) digest of A6 and A6(dLORF11) cosmid DNAs with BamHI, EcoRI and HindIII showed expected RE patterns thus confirming the absence of rearrangement. The role of the rMd5(dLORF11) virus was studied in vitro and in vivo. In vitro growth characteristics of the rMd5(dLORF11) viruses were similar to those of parental rMd5, showing that LORF11 is not essential for replication in vitro. In vivo studies of rMd5(dLORF11) viruses showed that the virus was impaired in replication in chickens and did not induced tumors. We conclude that LORF11 is essential for replication in chickens.