Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2010
Publication Date: 3/1/2011
Citation: Niikura, M., Kim, T., Silva, R.F., Dodgson, J., Cheng, H.H. 2011. Virulent Marek's disease virus generated from infectious bacterial artificial chromosome clones with complete DNA sequence and implication of viral genetic homogeneity in pathogenesis. Journal of General Virology. 92:598-607. Interpretive Summary: Marek’s disease (MD) is an economically-important disease of chickens caused by a pathogenic virus known as the Marek’s disease virus (MDV). While MD is controlled by vaccines, new and more virulent MDV strains continue to evolve and appear in the field, thus, it is of great importance to understand the function of viral genes, especially those that are involved in higher virulence or might aid in improved vaccines. In this study, we generate, characterize and completely sequence two infectious MDV clones. The major advantage is that these clones provide the basic building blocks for future studies on viral gene function and more since the sequence is known. Ultimately, this tool should help in the generation of new MD vaccines with improved protective abilities.
Technical Abstract: Genetic homogeneity of a test population is essential to precisely associate a viral genome sequence and its phenotype at the nucleotide level. However, homogeneity is not easy to achieve for Marek’s disease virus (MDV) due to its strictly cell-associated replication. To address this problem, two virulent infectious bacterial artificial chromosome (BAC) clones of MDV were generated from an MDV genome previously cloned as five overlapping cosmids. The Md5SN5BAC clone has the BAC vector inserted between the 3’ ends of UL3 and UL4, such that no known open reading frames should be disrupted. The BAC vector is flanked by loxP sites, so that it can be deleted from the viral genome by transfecting Md5SN5BAC into a newly developed chicken cell line that constitutively expresses Cre recombinase. The Md5B40BAC clone has the BAC vector replacing a portion of US2, a location similar to that used by other groups to construct MDV BAC clones. Although both BACs were capable of producing infectious virulent MDV, when inoculated into susceptible chickens, Md5B40BAC-derived viruses showed somewhat better replication in vivo and higher virulence. Removal of the BAC vector in Md5SN5BAC-derived viruses had no influence on virulence. Interestingly, when genetically homogeneous virulent MDV generated from Md5B40BAC was mixed with avirulrent virus, the overall virulence of the mixed population was compromised noticeably, which suggests the competitive nature of the virulent and avirulent MDV in a host and emphasizes the importance of MDV population complexity in pathogenesis.