Clustering of mutations within the inverted repeat regions of a serially passaged attenuated gallid herpesvirus type 2 strain.

Marek's disease (MD) is the leading cause of losses in chicken production in the world. Over the past 40 years significant progress has been made in the control of MD through the use of vaccines which reduce or delay tumor formation in vaccinated flocks. However, these vaccines fail to induce an immune response that protects against infection and virus shedding. Little is known about the genetic changes that lead to attenuation and are necessary for the generation of vaccine strains. Previous research has demonstrated that serial passage of virulent strains in cell culture results in the generation of attenuated progeny. Obtaining detailed knowledge of the changes which are needed for attenuation will be important for advancing our understanding of MD biology and should facilitate the development of more potent vaccines. We have determined the complete nucleotide sequence of a bacterial artificial chromosome (BAC) construct representing the 80th passage of a very virulent plus (vv+) MD virus strain termed 584A. Pathotyping studies have indicated that this strain (584Ap80) is indeed attenuated. Bioinformatic analysis of the sequencing data has identified numerous gross genetic changes clustering in the inverted repeat regions of the genome, as well as subtle changes (single nucleotide polymorphisms or SNPs) scattered throughout the genome. Relative to the parental strain (584Ap9), insertional mutations were identified in the MD-specific genes encoding RLORF1, RLORF3, RLORF6, 23 kDa, RLORF7 (Meq), vIL8, vLip, RSORF1, and five uncharacterized novel genes. Deletions were found in four locations within the 584Ap80 genome. A large deletion (297nt) was found in the diploid genes 85.6/98.6 and a 321 nt deletion within the intergenic region between the U(L)3 and U(L)3.5 genes is predicted to create a fusion polypeptide. A single nucleotide deletion was identified within the origin of replication. Both insertions and deletions were found in the dipoid genes MDV3.4/78.3 encoding the virulence factor RLORF4. The sequencing of the attenuated strain 584Ap80 and comparison to that of the virulent parent 584A passage 9 (584Ap9) has provided a wealth of information regarding genetic changes which have occurred during the attenuation process.