Comparative Analysis of Multiple Consensus Genomes of the Same Strain of Marek’s Disease Virus Reveals Intrastrain Variation

Abstract

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloguing divergent nucleotides between strains with different phenotypes. However, each MDV strain is typically represented by a single low-coverage consensus genome despite the confirmed existence of mixed viral populations. To assess the reliability of consensus-only interstrain comparisons, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform 3-way comparisons between consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 240 positions involving 12 open reading frames (ORFs). We found that Md5 genomes varied only in 12 intergenic positions, with no variation within ORFs. Phylogenomic analyses showed all three Md5 consensus genomes clustered closely together, while also showing that CVI988-GenBank diverged from CVI988-UK and CVI988-USDA. Comparison of CVI988 consensus genomes with those of seven closely related isolates from China revealed 19 SNPs that appear to be unique to CVI988-GenBank. Finally, we evaluated the genomic homogeneity of CVI988 and Md5 populations by identifying positions with >2% read support for alternative alleles in two ultra-deeply sequenced samples. We were able to confirm that both populations of CVI988 and Md5 were mixed, exhibiting a total of 30 and 27 minor variant positions, respectively. We found no evidence of minor variants in the positions corresponding to the 19 unique SNPs in CVI988-GenBank. Taken together, our findings challenge prior claims of natural recombination between CVI988 and virulent strains and confirm the need to resequence MDV strains in order to ensure the accuracy of interstrain comparisons.