Complete Genome Sequence of Herpes Simplex Virus 1 Strain MacIntyre.

Herpes simplex virus type 1 (HSV-1) strain MacIntyre has a severe defect in the anterograde spread after replication in the nucleus. To better understand and identify the genetic determinants that lead to the unique phenotypes of the MacIntyre strain, we sequenced its genome with PacBio single-molecule real-time sequencing technology and resolved the complete sequence.

ANNOUNCEMENT

Herpes simplex virus type 1 (HSV-1) is a member of the Alphaherpesvirinae subfamily of the Herpesviridae family and has a linear double-stranded DNA genome (1), which contains ∼80 genes. HSV-1 strain MacIntyre originated from the brain of a patient with fatal encephalitis (2–4). It egresses and infects only neurons that are presynaptic to the infected neuron (5–13).

We obtained the MacIntyre strain from Advanced Biotechnologies (Columbia, MD). The virus was propagated in Vero cells (ATCC, Gaithersburg, MD), and the viral stock was titrated using a plaque-forming assay (14). The genomic DNA was isolated by using a PureLink viral DNA minikit (Invitrogen, Carlsbad, CA) following the manufacturer’s protocols. Viral DNA shearing was carried out using an M220 sonicator (Covaris, Woburn, MA). Size selection and subsequent cleanup were performed using a combination of BluePippin (Sage Science, Beverly, MA) with the red miniTUBE (Covaris) and AMPure PB beads (PacBio, Menlo Park, CA), respectively. After shearing, bands ∼5 kb long were used for library construction using the SMRTbell template prep kit v1.0 (PacBio) with two alterations, an A-tailing step as well as overhang adapter ligation, as opposed to blunt adapter ligation. The constructed libraries were then sequenced using PacBio long-read sequencing on a Sequel system using v3.0 chemistry with a 6 pM loading concentration and a 10-h runtime.

A total of 4,230,138 subreads (mean length, 2,344 bp; maximum length, 22,799 bp; total number of bases, 9,917,428,361) from the 5′ end of the genome to the 3′ end were assembled into two contigs 126,677 and 25,074 bases long by using the PacBio de novo assembler Hierarchical Genome Assembly Process v4.0 (HGAP4) (15) (default settings were used, except that the seed length cutoff was set to 15 kbp, and the minimum required alignment length was set to 500 bp). We aligned the two contigs against the genome sequence of HSV-1 reference strain 17 (GenBank accession number JN555585) (16) and found a gap of ∼600 bp between them in the repeat long (RL) region of strain 17. The two contigs were ordered and assembled into a draft sequence; the gap was filled by aligning all the reads to the draft sequence, and consensus was called by using the genome resequencing pipeline of SMRT Analysis v6.0 with default settings (17). The average depth per base pair over the final resolved genome sequence is 61,410 reads, a coverage that ensures high-quality consensus base calling (>99.99% accuracy) (15). The genome termini were determined by a short sequence, a, which is repeated at both ends of the genome and at the L-S junction (18). The a sequence (473 bp) uncovered by this new assembly has unique features in its DR2 region compared to those of other HSV-1 strains that have been reported (19).

The finished length of the complete genome is 152,476 bases with a GC content of 68.21%. It resolves ∼5.8 kbp of undetermined regions in the existing partial genome sequence (GenBank accession number KM222720) (20), with gaps closed in the genes LAT, RL1, RL2, RS1, UL36, US1, US2, US9, and US12. It also confirmed that three genes, UL46, US7, and US9, contain new stop-codon positions (previously reported under GenBank accession number KM222720) due to homopolymer-based frameshifts or single nucleotide variations. These variations may contribute to the unique phenotype of MacIntyre, but further experiments need to be conducted to associate observed differences with phenotype effects.

The whole-genome nucleotide similarity between the new genome and HSV-1 strain 17 is 97.48% based on BLASR (v2.0.0) alignment (21) with default parameters. The annotations for genes and coding sequences were transferred from HSV-1 strain 17 to the MacIntyre strain based on sequence homology (22, 23).

Data availability.

This genome sequence was deposited at GenBank under the accession number MN136523, with SRA accession number SRR9719185.