Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions

Abstract

Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot. IMPORTANCEHerpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genomewide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large inverted repeats constitute a recombination hot spot. Additionally, Monte Carlo analyses found biases toward high GC content and intergenic and repetitive regions. Herpes simplex virus 1 (HSV-1) is a double-stranded DNA (dsDNA) virus in the Alphaherpesvirinae subfamily which causes recurrent, mucocutaneous lesions. HSV-1 is the leading cause of both sporadic encephalitis and infectious keratitis in the United States (1, 2). Animal studies have shown that disease severity is dependent on three factors: innate host resistance, the host immune response, and the viral strain (1, 3-16). Previous work of ours examining the role of the viral strain in virulence involved generating recombinant HSV-1 strains through mixed infections with two strains, OD4 and CJ994 (17). The advent of next-generation sequencing technologies has allowed multiple HSV-1 genomes to be sequenced (18,19), thus allowing the opportunity to sequence previously generated recombinants and examine genome-level recombination phenomena.The HSV-1 genome is approximately 152 kb in length and is arra...

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“…The OD4 and CJ994 strains were low-passage-number, clinically derived, plaque-purified isolates from Seattle, WA, USA, and were characterized previously (15). The OD4-CJ994 recombinants were generated using both in vivo and in vitro methods (15,40). Four of the 21 in vivo-derived recombinants were newly generated for the current study and have not been previously described (Table 1).…”

“…The genomic sequencing and assembly of 40 OD4-CJ994 recombinants out of the 65 HSV-1 recombinants in the current study have been previously described (40). The 25 new HSV-1 recombinants were sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) similarly to the previous approach (16).…”

“…The selected plaques were subjected to two additional rounds of plaque purification. Individual plaques (one per mouse to avoid isolating siblings) were picked, and high-titer stocks were prepared in Vero cells, as we have described previously (40).…”

“…The clarified supernatants (400 ϫ g; 10 min) were then serially diluted and plaqued onto Vero cells in six-well plates. Individual plaques were randomly picked and further plaque purified an additional two times, and high-titer stocks were prepared as described previously (40).…”

“…The number of breakpoints detected per genome varied, ranging from one breakpoint detected in recombinants 914-B and 914-E3 to 12 crossover events in recombinant IV-7. The breakpoint maps for the 25 newly sequenced recombinants can be referenced to the 40 preceding recombinants by accessing our previous work (40).…”