Molecular Evolution of Herpes Simplex Virus 2 Complete Genomes: Comparison between Primary and Recurrent Infections

Abstract

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates.IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine.

Keywords: Herpevac Trial for Women; complete genome; evolutionary biology; genetic polymorphisms; herpes simplex virus; next-generation sequencing.

FIG 1

Flow chart summarizing the data generation and analysis workflow. (1) HSV-2 was isolated from primary and recurrent infections in two subjects, and the viruses were expanded a single time in Vero cells. (2) Illumina Hi-Seq sequencing was performed on the whole genome of each sample. (3) De novo assembly of contigs was carried out using Velvet, and the contigs were aligned with the HG52 reference strain using Abacas/Nucmer to establish draft genome coverage rates. (4) Programmatic Novoalign and Bowtie2 alignments (BAM files), which excluded the IR_L and IR_S regions and used the HG52 strain as a reference, were compared based on the numbers of SNVs and indels that appeared in ≥10% of the Illumina read depth. Sanger sequencing was then performed on the HSV-2 regions with low read coverage (read depth, <25) based on Novoalign alignments. Sanger plus Novoalign consensus sequences for each sample (FASTA files) were aligned using nucleic and amino acid sequences. The HG52 and SD90e strains were used independently as reference genomes. Using the IUPAC code, SNVs observed in Novoalign alignments (≥10% of the read depth) were incorporated into the manually adjusted alignment of each sample. The BAM and FASTA files were uploaded to GenBank (Table 3). The rectangular and rounded boxes represent, respectively, databases and actions done in the databases.

FIG 2

Features of the complete HSV-2 genomes aligned with the HSV-2 strain HG52 reference sequence. Assembled Illumina mapped reads were aligned with the reference sequence and viewed in the IGV program. The scale at the top represents the genome position in kilobases. The horizontal bars below the genome scale represent the locations of the IR_L regions (light brown), U_L region (light pink), IR_S regions (orange), and U_S region (light blue) in the genome of HSV-2 reference strain HG52. Samples are labeled on the left. BAM file reads were capped at 100 for visualization in IGV, and the read depth at each nucleotide position is represented in dark gray for each sample (obtained from IGV). The vertical colored lines inserted in the gray section represent polymorphisms in the genome using the HG52 strain as a reference. HSV-2 coding regions are shown as green arrows indicating the direction of the ORF. The red arrows represent the locations of forward and reverse primers used in Sanger sequencing (Table 7) to fill the regions of low read depth coverage.

FIG 3

Number of HSV-2 genes at each interval of nonsynonymous change. The intervals (y axis) were selected based on the average percentage of SNVs that represented nonsynonymous changes across ORFs, using the HG52 strain as the reference sequence. The most polymorphic genes are identified. See Table S1 in the supplemental material for a list of all the SNVs observed in the ORFs of the complete HSV-2 genomes.

FIG 4

Features of the amino acid variants resulting from nonsynonymous changes in the primary or recurrent isolates of the tegument protein U_L37. Assessment of the tertiary molecular structure of pUL37 is shown (with structural modifications) based on the crystal structure of the N-terminal half of pseudorabiesvirus pUL37 (PDB accession no. 4K70; residues 24 to 570). Domains are numbered and represented in different colors. The polymorphic amino acids (P493H) (Table 6) are boxed and colored in cyan on the protein tertiary structures.

FIG 5

Bayesian majority rule consensus tree of 23 genomic HSV-2 sequences. The North American strains were from Kolb et al. (31) (red) and Newman et al. (30) (blue). The four HSV-2 complete genomes sequenced in this research are in black. (A) Complete Bayesian tree using HSV-1 and ChHV-1 as outgroups. (B) Expansion of the HSV-2-specific node presenting the Bayesian posterior probabilities (>90%) and maximum-parsimony bootstrap (100 replicates; >75%)/maximum-likelihood bootstrap (1,000 replicates; >75%) values above and below the branches, respectively.