Accumulation of attenuating mutations in varying proportions within a high passage very virulent plus strain of Gallid herpesvirus type 2

Marek's disease (MD) is controlled through mass vaccination. Although these vaccines reduce or delay tumor formation they fail to induce sterilizing immunity and prevent virus shedding. Relatively little is known about the genetic changes that lead to attenuation. It has been established that serial passage of virulent strains in avian cell lines results in the generation of attenuated progeny at some undefined passage level. A detailed cataloging of the mutations needed for attenuation will be important for advancing our understanding of MD biology and should facilitate the development of better vaccines. Using deep sequencing, the complete nucleotide sequence of the very virulent plus (vv+) strain 648A representing the 101st passage was determined. Pathotyping studies have indicated that 648Ap101 is indeed attenuated. Comparative sequence analysis with the 648A strain at passage 11 has identified numerous gross genetic changes and subtle single nucleotide polymorphisms scattered throughout the genome. Relative to the strain (648Ap11), deletions were identified in MD-specific genes located in the repeat long (RL), unique long (UL) and repeat short (RS) regions. A deletion in the RL region, present in 33% of the p101 sequences, mapped to the genes encoding viral interleukin 8 (vIL8), RLORF4 and RLORF5. An RS deletion was mapped to the Meq oncoprotein binding site within the ICP4 promoter and was present in 97.8% of the p101 sequences. A short deletion in the UL region mapped to the 3′ terminus of the gene encoding vLipase and was present in 54% of the p101 sequences. The cis-acting sites involved in DNA replication and packaging also contained deletions in varying proportions (64% and 100%, respectively). Three mutations, present in 100% of p101 sequences, were identified in the overlapping genes encoding the Arg-rich protein (MDV002/079) and the virus-encoding telomerase (vTR). Varying proportions of 12 single nucleotide polymorphisms (SNPs) were identified within 11 open reading frames (ORFs) and 3 noncoding regions. This comparative sequencing study has provided a wealth of information regarding genetic changes which have occurred during the attenuation process and has indicated that serial passage results in the generation of mixed populations.