Rapid Genome Assembly and Comparison Decode Intrastrain Variation in Human Alphaherpesviruses

Abstract

Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies.

Shared ancestry of herpes simplex virus 1 strain Patton with recent clinical isolates from Asia and with strain KOS63

“…Sequencing of GFP-US11 was performed by NYU Langone Medical Center Genome Technology Center (GTC), whereas the wild type virus was sequenced by Aperiomics (Ashburn, VA). For Patton GFP-US11, the sequence was assembled using VirGA (version 1.0), a galaxy-based viral genome assembly workflow (Parsons et al, 2015; Wan et al, 2015) provided on-line and free of charge by Moriah Szpara and colleagues (Pennsylvania State University). In brief, after trimming to remove redundant and repetitive sequences, the remaining short reads were assembled into longer contigs that could be orientated and joined into a draft genome using a reference-guided assembler and a scaffold based contig extender tool.…”

“…Some viruses exhibit larger deletions and frame shifts that may be deleterious for growth in vivo , however, for the most part individual strains appear relatively stable even after repeated passage in culture (Colgrove et al, 2015). Using sequence variation, individual HSV-1 strains can be grouped into three major clades or phylogroups of African, Asian and European/North American origin (Bowden et al, 2006; Parsons et al, 2015; Szpara et al, 2014; 2010). This diversity is thought to reflect the extended co-evolution of HSV-1 with humans and for the most part the distribution of genotypes mirrors the migration of our species across the globe (Bowden et al, 2006; Sakaoka et al, 1994).…”

Genotypic and phenotypic diversity within the neonatal HSV-2 population

“…Viral nucleocapsid gDNA was sheared using a Covaris M220 sonicator/disruptor under the following conditions: 60s duration, peak power 50, 10% duty cycle, at 4°C. Barcoded sequencing libraries were prepared using the Illumina TruSeq low-throughput protocol according to manufacturer's specifications and as previously described (75,76). The quality of sequencing libraries was evaluated by Qubit (Invitrogen, CA), Bioanalyzer (Agilent), peer-reviewed) is the author/funder.…”

“…A consensus genome was assembled for each viral isolate using a previously described Viral Genome Assembly (VirGA) bioinformatics workflow (75). VirGA begins by quality-filtering the MiSeq sequence reads and removing sequences that match the host (human) genome.…”

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument

“…(The CATC designation of this density is in line with the original gene annotations and functional analyses of pUL17, pUL25 and pUL36 as tegument proteins [32][33][34][35], and is chosen here to facilitate our comparison of tegument densities across subfamilies of herpesviruses.) By contrast, bherpesviruses possess a capsid-associated tegument protein, pp150, that forms a proteinaceous net enclosing the entire capsid, presumably to buttress it against the internal pressure of their large genomes [36][37][38][39][40].…”