Multiplex sequencing of seven ocular herpes simplex virus type-1 genomes: phylogeny, sequence variability, and SNP distribution

Abstract

Purpose: Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains.

Methods: A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot).

Results: Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions.

Conclusions: Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure-function studies.

Figure 1.

Schematic summarizing the mean peak ocular disease scores for blepharitis, stromal keratitis, neovascularization, and percent mortality of 4- to 6-week-old Balb/c mice infected with viral strains TFT401, CJ311, CJ360, CJ394, CJ970, and OD4. The scoring system and virulence data were presented in a previous publication. The virulence characteristics of 134 have not yet been determined.

Figure 2.

Coverage of multiplexed, sequenced, and assembled HSV-1 genomes. strain 17 was used as the reference sequence to assemble each of the genomes. (A) HSV-1 genome structure. (B) The percentage of GC content for the strain 17 genome. (C) Depth of coverage for each of the HSV-1 genomes sequenced. Pink shading: depth of coverage in a given area of the genome. (D) Depth of coverage versus GC content percentage.

Figure 3.

Whole genome phylogenetic analysis of multiple HSV-1 viral strains. The HSV-1 genomes analyzed include the seven ocular isolates sequenced in this work, as well as the previously published genomes of strains 17, F, and H129. (A) Consensus bootstrap neighbor-joining tree using HSV-2 strain HG52 as an outgroup. (B) Expansion of the HSV-1 specific node from the neighbor-joining tree in (A). The genomes were aligned with ClustalW and then consensus bootstrap (1000 replicates) neighbor-joining trees, using the Tamura-Nei algorithm, were generated with the Mega4 package. The phylogenetic distance is located at the bottom of each tree.

Figure 4.

Global sequence comparison of multiple HSV-1 genomes compared with the reference strain 17. The comparison plots were generated in mVista LAGAN (http://genome.lbl.gov/vista/index.shtml). Top: HSV-1 genome map. The genomic nucleotide positions using HSV-1 strain 17 as a reference are located on the x-axis. Red lines: manually applied indicators of sequencing gaps greater than 20 bp, catalogued in Supplementary Table S1 (http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7812/-/DCSupplemental).

Figure 5.

SNP distribution across the genomes. The annotated HSV-1 genome is shown across the top and the SNPs for each strain (using strain 17 as the reference) are shown below with each tick mark indicating an SNP. The repeat regions were not plotted because of lower coverage.

Figure 6.

Amino acid variation in proteins along the genome from multiple HSV-1 strains. (A) The average number of amino acid substitutions for each protein in HSV-1 strains TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4 compared to strain 17. (B) The average number of amino acid changes normalized to protein length. The mean, one standard deviation, and two standard deviations for each graph have been plotted. The name of each protein is located on the x-axis. The γ₁34.5, ICP0 and ICP4 proteins were not included, because of low sequence coverage. The reported numbers do not reflect the frameshift mutations found in UL2, UL13, UL17, UL42, and UL55, so as not to skew the data.

Figure 7.

Similarity plots of selected areas of the HSV-1 genome compared to strain 17. Areas of the genome featuring genes with variance greater than two standard deviations from Figure 4 (UL1, UL11, UL43, UL49A, US4, and US7) were analyzed with Simplot. The analysis includes plots of HSV-1 strains TFT401, 134, CJ311, CJ360, CJ394, CJ970, OD4, F, and H129. The UL1 (A), UL11 (B), UL43 (C), UL49A (C), US4 (D), and US7 (D) genes are highlighted in red.

Figure 8.

Consensus bootstrap neighbor-joining trees of selected α, β, and γ proteins. The nucleotide sequences of ICP27 (UL53), ICP47 (US12), UL42, ICP8 (UL29), glycoprotein L (UL1), and VP5 (UL19) from HSV-1 strains 17, F, H129, TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4 were each translated into their inferred amino acid sequences and then aligned with ClustalW. Consensus bootstrap (1000 replicates) neighbor-joining trees were then generated. The relative phylogenetic distance marker is located near the bottom of each tree.

Figure 9.

High variability proteins showing the locations of sequence differences using strain 17 as the baseline. UL49.5 is not shown, although it was identified as having higher variability.