Embracing Complexity: What Novel Sequencing Methods Are Teaching Us About Herpesvirus Genomic Diversity.

The arrival of novel sequencing technologies throughout the past two decades has led to a paradigm shift in our understanding of herpesvirus genomic diversity. Previously, herpesviruses were thought to exist as a family of DNA viruses with mostly identical genomes that evolved at much slower rates than RNA viruses. However, a growing body of evidence now suggests that herpesviruses exist as dynamic populations that possess standing variation and evolve at much faster rates than previously assumed. In this review, we explore how strategies such as deep sequencing, long-read sequencing, and haplotype reconstruction are allowing scientists to dissect the genomic composition of herpesvirus populations. We also discuss the challenges that need to be addressed before a detailed picture of herpesvirus diversity can emerge.

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

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 consensus genome despite the confirmed existence of mixed viral populations. To assess the reliability of single-consensus interstrain genomic 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 236 positions involving 13 open reading frames (ORFs). In contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Phylogenomic analyses showed all three Md5 consensus genomes clustered closely together, while also showing that CVI988 GenBank.BAC diverged from CVI988 Pirbright.lab and CVI988 USDA.PA.field . Comparison of CVI988 consensus genomes revealed 19 SNPs in the unique regions of CVI988 GenBank.BAC that were not present in either CVI988 Pirbright.lab or CVI988 USDA.PA.field . Finally, we evaluated the genomic heterogeneity 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 29 and 27 high-confidence minor variant positions, respectively. We did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988 GenBank.BAC . Taken together, our findings confirm that consensus genomes of the same strain of MDV can vary and suggest that multiple consensus genomes per strain are needed in order to maximize the accuracy of interstrain genomic comparisons.